Epigenetic alterations as mechanism of imatinib resistance in a cellular model of chronic myeloid leukemia

La CML è una patologia mieloproliferativa risultante dall’espansione policlonale di cellule staminali Il trattamento d’elezione è imatinib (IM). Nonostante il successo di IM nel giro di 18-24 mesi, circa il 30% dei pazienti sviluppa resistenza secondaria. Lo scopo del mio progetto è stato quello di indagare i possibili meccanismi genetici ed epigenetici (come la deregolazione dei miRNA e la metilazione aberrante del DNA) per determinare in che modo possano contribuire all’insorgenza di resistenza. Sono state allestite culture cellulari di K562 resistenti ad IM (da 0,05 fino a 3 µM). I miRNA sono stati analizzati per identificare un profilo caratteristico del processo di resistenza, mentre per gli mRNA sono state ricercate alterazioni nell’espressione dei geni addetti al trasporto dei farmaci. Per quanto riguarda il DNA, è stato valutato come variano i livelli di metilazione durante il processo di acquisizione della resistenza analizzando oltre 850.000 siti CpG. Dall’analisi dell’espressione dei trasportatori dei farmaci, è emerso che geni della famiglia dei trasportatori ABC sono sovraespressi nelle cellule resistenti. Per quanto riguarda i miRNA, è emerso che 6 miRNA sono significativamente deregolati: miR-193b-3p, miR-486-5p, miR-512-3p, miR-517a-3p, miR-365a-3p, miR-372-3p. Questi modulano geni appartenenti al pathway di segnalazione di ErbB e PI3K/Akt, coinvolti nei processi di vitalità cellulare, apoptosi, metabolismo e tumorigenesi. Per quanto riguarda la metilazione è stato osservato che, con l’incremento della dose somministrata, il numero di geni metilati aumenta notevolmente e, che in particolare, i geni PTPRF, TP73, ARHGEF10, FHDC1, DUSP6, PLD6 e MIR548H4 sono significativamente ipermetilati nelle cellule resistenti. Conclusioni. Data la recente attenzione rivolta verso il ruolo dei meccanismi epigenetici nell’insorgenza di resistenza, è possibile che un profiling genetico ed epigenetico, che tenga conto di come interagiscono fra loro i trasportatori di efflusso, i miRNA e la metilazione del DNA, possa rappresentare una svolta per la terapia mirata.CML is a myeloproliferative disorder resulting from polyclonal stem cell expansion. The standard treatment is imatinib (IM). Despite the success of IM, within 18-24 months about 30% of patients develop secondary resistance. The aim of my project was to investigate possible genetic and epigenetic mechanisms (as the deregulation of miRNAs and aberrant DNA methylation), to determine how they can contribute to the resistance mechanism. Cell cultures of K562 resistant to IM (0.05 -3 μM) were set up. MiRNA, RNA and DNA were isolated. The miRNAs were analysed with a preformed tool to identify a profiling of the resistance process, while for mRNA alterations in the expression of the genes involved in the transport of drugs were sought. Regarding DNA, we analysed how methylation levels vary during the development of resistance by analyzing over 850,000 CpG sites. From the analysis of the drug transporters, it has emerged that many of the superfamily genes of the ABC transporters are overexpressed in the cells that have acquired resistance. Among these, worthy of note are ABCG2, ABCA3 and ABCC1. Comparing the miRNA expressions to the different concentrations with untreated, we observed that 6 miRNAs are significantly deregulated: miR-193b-3p, miR-486-5p, miR-512-3p, miR-517a-3p, miR-365a -3p, miR-372-3p. These miRNAs modulate genes belonging to the ErbB signaling pathway, involved in the processes of modulation of cell viability, apoptosis, and tumorigenesis mechanism. Regarding methylation, it has been observed that the number of methylated genes increases considerably and the PTPRF, TP73, ARHGEF10, FHDC1, DUSP6, PLD6 and MIR548H4 genes are significantly hypermethylated in resistant cells. Given the recent attention to the role of epigenetic mechanisms in the onset of resistance, it is possible that a genetic and epigenetic profiling, which takes into account how the efflux transporters, miRNAs and DNA methylation interact, can represent a carried out for target therapy

Personalized Medicine in Gastrointestinal Stromal Tumor (GIST): Clinical Implications of the Somatic and Germline DNA Analysis

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. They are characterized by gain of function mutations in KIT or PDGFRA tyrosine kinase receptors, with their consequent constitutive activation. The gold standard therapy is imatinib that offers a good and stable response for approximately 18-36 months. However, resistance is very common and it is vital to identify new biomarkers. Up until now, there have been two main approaches with focus to characterize novel targets. On the one hand, the focus is on the tumor genome, as the final clinical outcome depends mainly from the cancer specific mutations/alterations patterns. However, the germline DNA is important as well, and it is inconceivable to think the patients response to the drug is not related to it. Therefore the aim of this review is to outline the state of the art of the personalized medicine in GIST taking into account both the tumor DNA (somatic) and the patient DNA (germline)

Mechanisms of resistance to a PI3K inhibitor in gastrointestinal stromal tumors: an omic approach to identify novel druggable targets

Background: Gastrointestinal stromal tumors (GISTs) represent a worldwide paradigm of target therapy. The introduction of tyrosine kinase inhibitors has deeply changed the prognosis of GIST patients, however, the majority of them acquire secondary mutations and progress. Unfortunately, besides tyrosine-kinase inhibitors, no other therapeutic options are available. Therefore, it is mandatory to identify novel molecules and/or strategies to overcome the inevitable resistance. In this context, after promising preclinical data on the novel PI3K inhibitor BYL719, the NCT01735968 trial in GIST patients who had previously failed treatment with imatinib and sunitinib started. BYL719 has attracted our attention, and we comprehensively characterized genomic and transcriptomic changes taking place during resistance. Methods: For this purpose, we generated two in vitro GIST models of acquired resistance to BYL719 and performed an omic-based analysis by integrating RNA-sequencing, miRNA, and methylation profiles in sensitive and resistant cells. Results: We identified novel epigenomic mechanisms of pharmacological resistance in GISTs suggesting the existence of pathways involved in drug resistance and alternatively acquired mutations. Therefore, epigenomics should be taken into account as an alternative adaptive mechanism. Conclusion: Despite the fact that currently we do not have patients in treatment with BYL719 to verify this hypothesis, the most intriguing result is the involvement of H19 and PSTA1 in GIST resistance, which might represent druggable targets

Izostanak mutacija humanoga interferon alfa-2b gena u radnika kronično izloženih ionizirajućem zračenju

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure.Kronična izloženost niskim razinama ionizirajućega zračenja povezana je s rizikom od dugoročnih štetnih posljedica za zdravlje, što obuhvaća i mutacije gena te nastanak raka. U tijeku je potraga za pouzdanim biopokazateljima izloženosti ionizirajućem zračenju u ljudi, budući da njihova primjena može značajno unaprijediti praćenje profesionalno izloženih osoba. U tom smislu, a s obzirom na ranija saznanja, cilj je ovoga istraživanja bio utvrditi mutacije gena za proizvodnju humanoga interferona alfa-2b (hIFNα-2b gena) kao mogućega biopokazatelja profesionalne kronične izloženosti niskim dozama ionizirajućega zračenja, koje je usto povezano s djelovanjem na hematopoezu i pad imuniteta. Analiziran je genomski DNA 51 rudara u rudnicima uranija te 38 kontrolnih ispitanika iz Kazahstana, odnosno genomski DNA 21 zdravstvenoga radnika na radiologiji i 21 kontrolnoga ispitanika iz Italije. Mutacije hIFNα-2b gena utvrđivane su metodom lančane reakcije polimerazom u stvarnom vremenu (engl. real-time PCR) odnosno sekvenciranjem prema Sangeru, ali se pokazalo da niti jedan radnik nije imao niti jednu od deset traženih mutacija toga gena. Stoga ne preostaje drugo nego i dalje tražiti pouzdane biopokazatelje za rano otkrivanje štetnih zdravstvenih učinaka povezanih s kroničnom izloženosti niskim dozama ionizirajućega zračenja

Pharmacogenetics of tyrosine kinase inhibitors in gastrointestinal stromal tumor and chronic myeloid leukemia

Introduction: Gastrointestinal stromal tumors (GIST) and chronic myeloid leukemia (CML) are two tumor types deeply different from each other. Despite the differences, these disorders share treatment with tyrosine kinase inhibitor imatinib. Despite the success of imatinib, the response rates vary among different individuals and pharmacogenetics may play an important role in the final clinical outcome. Areas covered: In this review, the authors provide an overview of the pharmacogenetic literature analyzing the role of polymorphisms in both GIST and CML treatment efficacy and toxicity. Expert opinion: So far, several polymorphisms influencing the pharmacokinetic determinants of imatinib have been identified. However, the data are not yet conclusive enough to translate pharmacogenetic tests in clinical practice. In this context, the major obstacles to pharmacogenetic test validation are represented by the small sample size of most studies, ethnicity and population admixture as confounding source, and uncertainty related to genetic variants analyzed. In conclusion, a combination of different theoretical approaches, experimental model systems and statistical methods is clearly needed, in order to appreciate pharmacogenetics applied to clinical practice in the near future

Key Genetic and Epigenetic Mechanisms in Chemical Carcinogenesis

DNA sequence and genetic factors alone cannot fully explain the many processes implicated in diseases initiation and development. It is now well understood that additional factors are involved in a final resulting phenotype. Epigenetic modifications, heritable changes not affecting the DNA sequence, are a key phenomenon at the basis of normal growth and differentiation. However, these can be defective leading to diseases, such as cancer. An increasing body of literature reports the environmental and occupational exposure to a mixture of natural and man-produced substances leading to epigenetic alterations. The identification of key genetic and/or epigenetic events involved in chemical carcinogenesis is an important step towards the discovery of biomarkers that can be used to evaluate the exposure, predict biological effects, and prevent adverse health consequences. Here, we focus on epidemiological studies to review the most recent advances in understanding genetic and epigenetic factors in relation to particulate matter, benzene and polycyclic aromatic hydrocarbons exposure.DNA sequence and genetic factors alone cannot fully explain the many processes implicated in diseases initiation and development. It is now well understood that additional factors are involved in a final resulting phenotype. Epigenetic modifications, heritable changes not affecting the DNA sequence, are a key phenomenon at the basis of normal growth and differentiation. However, these can be defective leading to diseases, such as cancer. An increasing body of literature reports the environmental and occupational exposure to a mixture of natural and man-produced substances leading to epigenetic alterations. The identification of key genetic and/or epigenetic events involved in chemical carcinogenesis is an important step towards the discovery of biomarkers that can be used to evaluate the exposure, predict biological effects, and prevent adverse health consequences. Here, we focus on epidemiological studies to review the most recent advances in understanding genetic and epigenetic factors in relation to particulate matter, benzene and polycyclic aromatic hydrocarbons exposure. \ua9 The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved

Application of the lymphocyte Cytokinesis-Block Micronucleus Assay to populations exposed to petroleum and its derivatives: Results from a systematic review and meta-analysis

The lymphocyte cytokinesis-block micronucleus (CBMN) assay is applied in many different in vivo biomonitoring studies of human exposure to genotoxic chemicals. Among extensively chemicals investigated, we identified petroleum and its derivatives, in particular benzene and the most common mixture of benzene, toluene, and xylene. Although conflicting results have been reported on the effects of benzene exposure, the number of positive findings in independent studies suggests that occupational exposure to benzene causes DNA damage in peripheral blood lymphocytes. To assess current evidence on this hypothesis, we conducted a meta-analysis. Our aim was to evaluate the effect of benzene exposure on genetic damage, quantified using the CBMN assay on individuals occupationally exposed to petroleum and its derivatives. Statistical analyses were conducted using the rmeta package from the free Software Environment for Statistical Computing R. Combined study results indicated that benzene exposure is associated with an increased level of genetic damage in peripheral blood lymphocytes, as reflected by an increased MN frequency. The summary mean difference in MN frequency between exposed and unexposed individuals was 1.64 (95% CI: 0.80-2.47). Overall, this finding points to MN frequency as a sensitive biomarker which could be used to evaluate genetic damage induced by occupational - industrial or environmental - exposure to benzene. This review also identified some important knowledge gaps as well as the need of large, well-designed studies. In particular, it is fundamental to accurately characterize the investigated population, including dietary habits and genetic variability which could modulate MN frequency in both exposed individuals and unexposed controls. In conclusion, according to present findings the use of the CBMN assay in biomonitoring studies could provide objective evidence to guide prioritization of preventive interventions in subjects occupationally exposed to petroleum derivatives, and in particular benzene

Clinical relevance of circulating molecules in cancer: focus on gastrointestinal stromal tumors

In recent years, growing research interest has focused on the so-called liquid biopsy. A simple blood test offers access to a plethora of information, which might be extremely helpful in understanding or characterizing specific diseases. Blood contains different molecules, of which circulating free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and extracellular vesicles (EVs) are the most relevant. Conceivably, these molecules have the potential for tumor diagnosis, monitoring tumor evolution, and evaluating treatment response and pharmacological resistance. This review aims to present a state-of-the-art of recent advances in circulating DNA and circulating RNA in gastrointestinal stromal tumors (GISTs). To date, progress in liquid biopsy has been scarce in GISTs due to several issues correlated with the nature of the pathology. Namely, heterogeneity in primary and secondary mutations in key driver genes has greatly slowed the development and application in GISTs, unlike in other tumor types in which liquid biopsy has already been translated into clinical practice. However, meaningful novel data have shown in recent years a significant clinical potential of ctDNA, CTCs, EVs and circulating RNA in GISTs

Gastrointestinal stromal tumors (GIST): Facing cell death between autophagy and apoptosis

Autophagy and apoptosis are 2 fundamental biological mechanisms that may cooperate or be antagonistic, although both are involved in deciding the fate of cells in physiological or pathological conditions. These 2 mechanisms coexist simultaneously in cells and share common upstream signals and stimuli. Autophagy and apoptosis play pivotal roles in cancer development. Autophagy plays a key function in maintaining tumor cell survival by providing energy during unfavorable metabolic conditions through its recycling mechanism, and supporting the high energy requirement for metabolism and growth. This review focuses on gastrointestinal stromal tumors and cell death through autophagy and apoptosis, taking into account the involvement of both of these processes in tumor development and growth and as mechanisms of drug resistance. We also focus on the crosstalk between autophagy and apoptosis as an emerging field with major implications for the development of novel therapeutic options

Prognostic Role of miR-221 and miR-222 Expression in Cancer Patients: A Systematic Review and Meta-Analysis

Background: A wealth of evidence has shown that microRNAs (miRNAs) can modulate specific genes, increasing our knowledge on the fine-tuning regulation of protein expression. miR-221 and miR-222 have been frequently identified as deregulated across different cancer types; however, their prognostic significance in cancer remains controversial. In view of these considerations, we performed an updated systematic review and meta-analysis of published data investigating the effects of miR-221/222 on overall survival (OS) and other secondary outcomes among cancer patients. A systematic search of PubMed, Web of Knowledge, and Cochrane Library databases was performed. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were used to assess the strength of association. Results: Fifty studies, analyzing 6086 patients, were included in the systematic review. Twenty-five studies for miR-221 and 17 studies for miR-222 which assessed OS were included in the meta-analysis. High expression of miR-221 and miR-222 significantly predicted poor OS (HR: 1.48, 95% CI: 1.14-1.93, p = 0.003 and HR: 1.90, 95% CI: 1.43-2.54, p < 0.001, respectively). Subgroup analysis revealed that the finding on miR-221 was not as robust as the one on miR-222. Furthermore, high miR-222 expression was also associated with worse progression-free survival and disease-free survival pooled with recurrence-free survival. Conclusions: The meta-analysis demonstrated that high expression of miR-222 is associated with poor prognosis in cancer patients, whereas the significance of miR-221 remains unclear. More work is required to fully elucidate the role of miR-221 and miR-222 in cancer prognosis, particularly in view of the limitations of existing results, including the significant heterogeneity and limited number of studies for some cancers