Radical chemistry of the catechol/quinone system and its role in the control of lipid peroxidation and melanin biosynthesis

The catechol (1,2-dihydroxybenzene) is a privileged structural motif among natural antioxidants like flavonoids, owing to its reactivity with alkylperoxyl radicals due to the stability of the semiquinone radical. The exploration of the relevance and mechanism of this non-conventional antioxidant chemistry in heterogenous biomimetic systems (aqueous micelles and unilamellar liposomes) is explored for the first time in Chapter 1. Results show antioxidant behaviour that surpasses that of nature’s premiere antioxidant α-tocopherol and relies on the cross-dismutation of alkylperoxyl and hydroperoxyl radicals at the water-lipid interface with regeneration of the catechol function from the oxidized quinone. The design and synthesis of new biomimetic catechol-type antioxidants by conjugation of thiols (e.g. cysteine) with quinones highlighted an unusual 1,6-type regioselectivity, which had been previously reported but never fully rationalized. Owing to its importance both in nature and in the development of new antioxidants, we investigated it in detail in Chapter 2. We could prove the onsetting of a radical-chain mechanism intermediated by thiyl and thiosemiquinone radicals at the basis of the “anomalous nucleophilic addition” of thiols to ortho-quinones, which paves the way to better understanding of the chemistry of such systems. The oxidation of catechols to the corresponding quinones is also a key reaction in the biosynthesis of melanins, mediated by enzyme Tyrosinase.

Genetica molecolare dell'autismo: studi di associazione ed analisi di geni candidati

Autism is a neurodevelpmental disorder characterized by impaired verbal communication, limited reciprocal social interaction, restricted interests and repetitive behaviours. Twin and family studies indicate a large genetic contribution to ASDs (Autism Spectrum Disorders). During my Ph.D. I have been involved in several projects in which I used different genetic approaches in order to identify susceptibility genes in autism on chromosomes 2, 7 and X: 1)High-density SNP association and CNV analysis of two Autism Susceptibility Loci. The International Molecular Genetic Study of Autism Consortium (IMGSAC) previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we evaluated the patterns of linkage disequilibrium (LD) and the distribution of haplotype blocks, utilising data from the HapMap project, across the two strongest peaks of linkage on chromosome 2 and 7. More than 3000 SNPs have been selected in each locus in all known genes, as well as SNPs in non-genic highly conserved sequences. All markers have been genotyped to perform a high-density association analysis and to explore copy number variation within these regions. The study sample consisted of 127 and 126 multiplex families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Association and CNV analysis implicated several new genes, including IMMP2L and DOCK4 on chromosome 7 and ZNF533 and NOSTRIN on the chromosome 2. Particularly, my contribution to this project focused on the characterization of the best candidate gene in each locus: On the AUTS5 locus I carried out a transcript study of ZNF533 in different human tissues to verify which isoforms and start exons were expressed. High transcript variability and a new exon, never described before, has been identified in this analysis. Furthermore, I selected 31 probands for the risk haplotype and performed a mutation screen of all known exons in order to identify novel coding variants associated to autism. On the AUTS1 locus a duplication was detected in one multiplex family that was transmitted from father to an affected son. This duplication interrupts two genes: IMMP2L and DOCK4 and warranted further analysis. Thus, I performed a screening of the cohort of IMGSAC collection (285 multiplex families), using a QMPSF assay (Quantitative Multiplex PCR of Short fluorescent Fragments) to analyse if CNVs in this genic region segregate with autism phenotype and compare their frequency with a sample of 475 UK controls. Evidence for a role of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family. 2)Analysis of X chromosome inactivation. Skewed X chromosome inactivation (XCI) is observed in females carrying gene mutations involved in several X-linked syndromes. We aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 164 affected girls. The study sample included families from different european consortia. I analysed the XCI inactivation pattern in a sample of italian mothers from singletons families with ASD and also a control groups (144 adult females and 40 young females). We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z score of 1.75 close to rs719489. In this region FMR1 and MECP2 have been associated in some cases with austim and therefore represent candidates for the disorder. I performed a mutation screen of MECP2 in 33 unrelated probands from IMGSAC and italian families, showing XCI skewness. Recently, Xq28 duplications including MECP2, have been identified in families with MR, with asymptomatic carrier females showing extreme (>85%) skewing of XCI. For these reason I used the sample of probands from X-skewed families to perform CNV analysis by Real-time quantitative PCR. No duplications have been found in our sample. I have also confirmed all data using as alternative method the MLPA assay (Multiplex Ligation dependent Probe Amplification). 3)ASMT as functional candidate gene for autism. Recently, a possible involvement of the acetylserotonin O-methyltransferase (ASMT) gene in susceptibility to ASDs has been reported: mutation screening of the ASMT gene in 250 individuals from the PARIS collection revealed several rare variants with a likely functional role; Moreover, significant association was reported for two SNPs (rs4446909 and rs5989681) located in one of the two alternative promoters of the gene. To further investigate these findings, I carried out a replication study using a sample of 263 affected individuals from the IMGSAC collection and 390 control individuals. Several rare mutations were identified, including the splice site mutation IVS5+2T>C and the L326F substitution previously reported by Melke et al (2007), but the same rare variants have been found also in control individuals in our study. Interestingly, a new R319X stop mutation was found in a single autism proband of Italian origin and is absent from the entire control sample. Furthermore, no replication has been found in our case-control study typing the SNPs on the ASMT promoter B.

Catalytic liquid- and gas-phase oxidations for the synthesis of intermediates and specialty chemicals: some examples of industrial relevance

Nowadays, it is clear that the target of creating a sustainable future for the next generations requires to re-think the industrial application of chemistry. It is also evident that more sustainable chemical processes may be economically convenient, in comparison with the conventional ones, because fewer by-products means lower costs for raw materials, for separation and for disposal treatments; but also it implies an increase of productivity and, as a consequence, smaller reactors can be used. In addition, an indirect gain could derive from the better public image of the company, marketing sustainable products or processes. In this context, oxidation reactions play a major role, being the tool for the production of huge quantities of chemical intermediates and specialties. Potentially, the impact of these productions on the environment could have been much worse than it is, if a continuous efforts hadn’t been spent to improve the technologies employed. Substantial technological innovations have driven the development of new catalytic systems, the improvement of reactions and process technologies, contributing to move the chemical industry in the direction of a more sustainable and ecological approach. The roadmap for the application of these concepts includes new synthetic strategies, alternative reactants, catalysts heterogenisation and innovative reactor configurations and process design. Actually, in order to implement all these ideas into real projects, the development of more efficient reactions is one primary target. Yield, selectivity and space-time yield are the right metrics for evaluating the reaction efficiency. In the case of catalytic selective oxidation, the control of selectivity has always been the principal issue, because the formation of total oxidation products (carbon oxides) is thermodynamically more favoured than the formation of the desired, partially oxidized compound. As a matter of fact, only in few oxidation reactions a total, or close to total, conversion is achieved, and usually the selectivity is limited by the formation of by-products or co-products, that often implies unfavourable process economics; moreover, sometimes the cost of the oxidant further penalizes the process. During my PhD work, I have investigated four reactions that are emblematic of the new approaches used in the chemical industry. In the Part A of my thesis, a new process aimed at a more sustainable production of menadione (vitamin K3) is described. The “greener” approach includes the use of hydrogen peroxide in place of chromate (from a stoichiometric oxidation to a catalytic oxidation), also avoiding the production of dangerous waste. Moreover, I have studied the possibility of using an heterogeneous catalytic system, able to efficiently activate hydrogen peroxide. Indeed, the overall process would be carried out in two different steps: the first is the methylation of 1-naphthol with methanol to yield 2-methyl-1-naphthol, the second one is the oxidation of the latter compound to menadione. The catalyst for this latter step, the reaction object of my investigation, consists of Nb2O5-SiO2 prepared with the sol-gel technique. The catalytic tests were first carried out under conditions that simulate the in-situ generation of hydrogen peroxide, that means using a low concentration of the oxidant. Then, experiments were carried out using higher hydrogen peroxide concentration. The study of the reaction mechanism was fundamental to get indications about the best operative conditions, and improve the selectivity to menadione. In the Part B, I explored the direct oxidation of benzene to phenol with hydrogen peroxide. The industrial process for phenol is the oxidation of cumene with oxygen, that also co-produces acetone. This can be considered a case of how economics could drive the sustainability issue; in fact, the new process allowing to obtain directly phenol, besides avoiding the co-production of acetone (a burden for phenol, because the market requirements for the two products are quite different), might be economically convenient with respect to the conventional process, if a high selectivity to phenol were obtained. Titanium silicalite-1 (TS-1) is the catalyst chosen for this reaction. Comparing the reactivity results obtained with some TS-1 samples having different chemical-physical properties, and analyzing in detail the effect of the more important reaction parameters, we could formulate some hypothesis concerning the reaction network and mechanism. Part C of my thesis deals with the hydroxylation of phenol to hydroquinone and catechol. This reaction is already industrially applied but, for economical reason, an improvement of the selectivity to the para di-hydroxilated compound and a decrease of the selectivity to the ortho isomer would be desirable. Also in this case, the catalyst used was the TS-1. The aim of my research was to find out a method to control the selectivity ratio between the two isomers, and finally to make the industrial process more flexible, in order to adapt the process performance in function of fluctuations of the market requirements. The reaction was carried out in both a batch stirred reactor and in a re-circulating fixed-bed reactor. In the first system, the effect of various reaction parameters on catalytic behaviour was investigated: type of solvent or co-solvent, and particle size. With the second reactor type, I investigated the possibility to use a continuous system, and the catalyst shaped in extrudates (instead of powder), in order to avoid the catalyst filtration step. Finally, part D deals with the study of a new process for the valorisation of glycerol, by means of transformation into valuable chemicals. This molecule is nowadays produced in big amount, being a co-product in biodiesel synthesis; therefore, it is considered a raw material from renewable resources (a bio-platform molecule). Initially, we tested the oxidation of glycerol in the liquid-phase, with hydrogen peroxide and TS-1. However, results achieved were not satisfactory. Then we investigated the gas-phase transformation of glycerol into acrylic acid, with the intermediate formation of acrolein; the latter can be obtained by dehydration of glycerol, and then can be oxidized into acrylic acid. Actually, the oxidation step from acrolein to acrylic acid is already optimized at an industrial level; therefore, we decided to investigate in depth the first step of the process. I studied the reactivity of heterogeneous acid catalysts based on sulphated zirconia. Tests were carried out both in aerobic and anaerobic conditions, in order to investigate the effect of oxygen on the catalyst deactivation rate (one main problem usually met in glycerol dehydration). Finally, I studied the reactivity of bifunctional systems, made of Keggin-type polyoxometalates, either alone or supported over sulphated zirconia, in this way combining the acid functionality (necessary for the dehydrative step) with the redox one (necessary for the oxidative step). In conclusion, during my PhD work I investigated reactions that apply the “green chemistry” rules and strategies; in particular, I studied new greener approaches for the synthesis of chemicals (Part A and Part B), the optimisation of reaction parameters to make the oxidation process more flexible (Part C), and the use of a bioplatform molecule for the synthesis of a chemical intermediate (Part D).

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

Isolamento, variabilità ed espressione del gene PIMT in Girasole

Il vigore è un aspetto rilevante della qualità delle sementi, strettamente connesso al loro status fisiologico, al genotipo e alle condizioni di stoccaggio. Stress ossidativi e danni alle macromolecole sono alla base del deterioramento del seme, che è equipaggiato con sistemi protettivi e di riparazione. Uno di questi coinvolge l’L-isoaspartil metiltransferasi (PIMT) che ripara i misfolding proteici catalizzando la riconversione in aspartato dell’isoaspartile anomalo accumulato. Scopo di questo studio era valutare il possibile ruolo del meccanismo di riparazione di PIMT nel vigore del seme in girasole. Per questo il relativo gene è stato isolato e caratterizzato, la variabilità allelica determinata su un campione di linee inbred e l’espressione genica misurata in risposta all’invecchiamento accelerato (aging) e al priming. La sequenza codificante ottenuta è costituita da 4 esoni e contiene i 5 domini caratteristici delle metiltransferasi. Il gene mostra elevata similarità con gli ortologhi vegetali e scarsa diversità nucleotidica nei genotipi coltivati rappresentativi della variabilità della specie. Nella sequenza aminoacidica, comunque, sono state rinvenute tre sostituzioni che potrebbero influenzare la funzionalità enzimatica. Dal punto di vista fisiologico i genotipi considerati hanno esibito notevole variabilità di risposte ai trattamenti, sia in termini di vigore che di espressione genica. Aging e priming hanno prodotto generalmente gli effetti attesi, rispettivamente negativi e positivi, sulla germinabilità e sulla sua velocità. In generale l’espressione di PIMT è risultata massima nel seme secco, come riportato altrove, e ridotta dall’aging. Anche il priming ha diminuito l’espressione rispetto al seme quiescente, mentre il suo effetto dopo l’aging è risultato genotipo-dipendente. Tuttavia, nelle condizioni descritte, non si sono evidenziate correlazioni significative tra vigore ed espressione di PIMT, tali da suggerire un chiaro ruolo di questo meccanismo nella qualità fisiologica del seme.

Fully bio-based epoxy resins

Epoxy resins are mainly produced by reacting bisphenol A with epichlorohydrin. Growing concerns about the negative health effects of bisphenol A are urging researchers to find alternatives. In this work diphenolic acid is suggested, as it derives from levulinic acid, obtained from renewable resources. Nevertheless, it is also synthesized from phenol, from fossil resources, which, in the current paper has been substituted by plant-based phenols. Two interesting derivatives were identified: diphenolic acid from catechol and from resorcinol. Epichlorohydrin on the other hand, is highly carcinogenic and volatile, leading to a tremendous risk of exposure. Thus, two approaches have been investigated and compared with epichlorohydrin. The resulting resins have been characterized to find an appropriate application, as epoxy are commonly used for a wide range of products, ranging from composite materials for boats to films for food cans. Self-curing capacity was observed for the resin deriving from diphenolic acid from catechol. The glycidyl ether of the diphenolic acid from resorcinol, a fully renewable compound, was cured in isothermal and non-isothermal tests tracked by DSC. Two aliphatic amines were used, namely 1,4-butanediamine and 1,6-hexamethylendiamine, in order to determine the effect of chain length on the curing of an epoxy-amine system and determine the kinetic parameters. The latter are crucial to plan any industrial application. Both diamines demonstrated superior properties compared to traditional bisphenol A-amine systems.

Epigenetic role of N-Myc in Neuroblastoma

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

New and More Sustainable Processes for the Synthesis of Phenolics: 2-phenoxyethanol and hydroxytyrosol

The research work has dealt with the study of new catalytic processes for the synthesis of fine chemicals belonging to the class of phenolics, namely 2-phenoxyethanol and hydroxytyrosol. The two synthetic procedures investigated have the advantages of being much closer to the Green Chemistry principles than those currently used industrially. In both cases, the challenge was that of finding catalysts and methods which led to the production of less waste, and used less hazardous chemicals, safer solvents, and reusable heterogeneous catalysts. In the case of 2-phenoxyethanol, the process investigated involves the use of ethylene carbonate (EC) as the reactant for phenol O-hydroxyethylation, in place of ethylene oxide. Besides being a safer reactant, the major advantage of using EC in the new synthesis is the better selectivity to the desired product achieved. Moreover, the solid catalyst based on Na-mordenite was fully recyclable. The reaction mechanism and the effect of the Si/Al ratio in the mordenite were investigated. In the case of hydroxytyrosol, which is one of the most powerful natural antioxidants, a new synthetic procedure was investigated; in fact, the method currently employed, the hydrolysis of oleuropein, an ester extracted from the waste water processing of the olive, makes use of large amounts of organic solvents (hexane, ethyl acetate), and involves several expensive steps of purification. The synthesis procedure set up involves first the reaction between catechol and 2,2-dimethoxyacetaldehyde, followed by the one-pot reduction of the intermediate to give the desired product. Both steps were optimized, in terms of catalyst used, and of reaction conditions, that allowed to reach ca 70% yield in each step. The reaction mechanism was investigated and elucidated. During a 3-month period spent at the University of Valencia (with Prof. A. Corma’s group), a process for the production of diesel additives (2,5-bis(propoxymethyl)furan) from fructose has been investigated.

Hierarchically organized chitin-based matrices

Chitin is the second most abundant biopolymer on Earth and the most diffused across the known species, being present in more than 70 % of them. It is present in a huge variety of different structures and morphologies being a massive pool of information for new material science approach. This thesis aims to study chitin at different level of organization using diverse approaches. Three main topics are discussed in this manuscript. The first is the use of a bottom-up approach to study chitin nanofibrils self-assembly in water triggering the assembly by pH increment. Successively, the assembly was studied in presence of another pH responsive biomacromolecule, the collagen, to get new composite materials and study how the assembly and the chitin/collagen ratio influence fibroblast’s viability. The second topic focuses on biogenic organized chitin-based matrices, in both Ariolimax californicus and Loligo vulgaris. This study aims to understand the features that give raise to the properties of those matrices. Finally, in the last section a top-up approach was used to exploit natural hierarchically organized chitinous matrices to obtain organized functional materials introducing a catechol on the free amino group of deacetylated chitin (DA 77 %). In conclusion, this study of chitin at different level of organization emphasized different novelties depending on the organization level studied. Moreover, this thesis gives many possibilities for future bioinspired routes to get highly organized materials, or for highly organized functional materials based on natural chitin-based matrices.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.

Role of CARM1 (PRMT4) in high grade serous ovarian cancer metabolism and function of PRMT5 in ARID1A- deficient endometrial cancer invasion

The arginine methyltransferase CARM1 (PRMT4) is amplified and overexpressed in ~20% of high-grade serous ovarian cancer (HGSOC) and correlates with a poor survival. Therapeutic approaches based on CARM1 expression remain to be an unmet need. Here we show that fatty acid metabolism represents a metabolic vulnerability for HGSOC in a CARM1 expression status dependent manner. CARM1 promotes the de novo synthesis of fatty acids and monounsaturated fatty acids (MUFAs). The disruption of MUFAs synthesis by inhibition of SCD1 results in excessive accumulation of cytotoxic saturated fatty acids and it is synthetic lethal with CARM1 expression. Collectively, our data show that the pharmacological inhibition of MUFAs synthesis via SCD1 inhibition represents a therapeutic strategy for CARM1-high HGSOC. Another arginine methyltransferase, PRMT5, has been identified by our CRISPR screening analysis as a promising candidate for invasive ARID1A-deficient endometrial cancer. Endometrial Cancer frequently harbor somatic inactivating mutation of ARID1A that can promote an invasive phenotype. Our in vitro approach validated the CRISPR screening showing that both PRTM5 knock down and its pharmaceutical inhibition specifically hamper the invasion of ARID1A inactivated cells. Mechanistically, PRMT5 directly regulates the epithelia to mesenchymal transition pathway genes interacting with the SWI/SNF complexes. Moreover, in vivo experiments showed that PRMT5 inhibition contrasted the myometrium invasion highlighting PRMT5 inhibition as promising therapeutic strategy for ARID1A- inactivated aggressive endometrial cancer.

Dissecting and resetting SETD2/H3K36ME3 deficiency in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of the BCR::ABL1 fusion gene, leading to a constitutively active tyrosine kinase that drives the disease. Genomic instability is a hallmark of CML, contributing to disease progression and treatment resistance. A study identified SETD2, a histone methyltransferase, as frequently dysfunctional in advanced-phase CML, resulting in reduced trimethylation of Histone H3 at lysine 36 (H3K36Me3). This loss is associated with poor prognosis and increased genetic instability. Investigations revealed that SETD2 dysfunction is caused by post-translational modifications mediated by Aurora kinase A and MDM2, leading to proteasome-mediated degradation. Aurora kinase A phosphorylates SETD2, while MDM2 ubiquitinates it, targeting it for degradation. Inhibition of MDM2 and Aurora kinase A restored SETD2 expression and activity, suggesting potential therapeutic targets. Loss of SETD2 and H3K36Me3 impairs DNA repair mechanisms, favoring error-prone repair pathways over faithful ones, exacerbating genetic instability. Reintroduction of SETD2 into deficient cells restored DNA repair pathways, preserving genomic integrity. Analysis of CD34+ progenitor cells from CML patients showed reduced SETD2 levels compared to healthy individuals, correlating with decreased clonogenic capacity. Notably, SETD2 loss is not detectable at diagnosis but emerges during disease progression, indicating its role as an early indicator of CML advancement. Therapeutically, inhibitors targeting Aurora kinase A, MDM2, and the proteasome showed efficacy in cells expressing SETD2, particularly in those with low SETD2 levels. Proteasome inhibitors induced apoptosis and DNA damage in SETD2-deficient cells, highlighting their potential for CML treatment. In conclusion, SETD2 acts as a tumor suppressor in CML, with its dysfunction contributing to genetic instability and disease progression. Targeting the mechanisms of SETD2 loss presents promising therapeutic avenues for controlling CML proliferation and restoring genomic integrity.