Role of epigenetically oriented therapy in the diffuse large B-cell lymphoma

Epigenetski orijentirana terapija potencijalno predstavlja novi način liječenja hematoloških novotvorina, uključujući i difuzni B-velikostanični limfom (DLBCL), pogotovo onih slučajeva koji su rezistentni na standardnu terapiju ili u bolesnika koji relapsiraju. U ovoj disertaciji pokazano je kako inhibitori histonske deacetilaze (HDACi) i hipometilirajuće tvari polučuju sinergistički učinak u zaustavljanju rasta malignih DLBCL stanica. Ovaj učinak je dokazan in vitro u analizi stanične vijabilnosti, indukcije apoptoze, aktivacije kaspaznog sustava te acetilacije histona računajući omjer relativnog rizika (RRR) u trajnim i primarnim limfomskim linijama. Učinkovitost panobinostata (LBH589) i decitabina u zaustavljanju rasta malignih stanica je potvrđena i u in vivo modelu ljudskog limfomskog ksenografta uz statistički značajnu razliku (p < 0.05) u prosječnom volumenu tumora na boku životinje u grupama koje su primile kombinaciju panobinostata i decitabina u odnosu na grupe životinja tretirane lijekom pojedinačno ili kontrolu koja je primila samo vektor. Mehanističko i molekularno objašnjenje sinergističkom učinku ove kombinacije istraženo je analizom metilacije genoma i promjene genske ekspresije tehnologijom mikropostroja kojima su identificirana tri potencijalna tumor supresora, TP73, MXD4 i CAV1, čija je aktivnost reaktivirana u limfomskim stanicama koje su tretirane panobinostatom i decitabinom. Spoznaje iz ove disertacije uključene su u širu analizu kako bi se stvorio model farmakologije i biologije sustava za B-stanične novotvorine, a praktično se primijenile u kliničkom istraživanju s epigenetski usmjerenom terapijom.Epigenetically targeted therapy potentially represents a novel way of treating hematologic malignancies, including diffuse large B-cell lymphoma (DLBCL), especially those cases resistant to standard therapeutic options and in patients with relapsed disease. As shown in this thesis, histone deacetylase inhibitors (HDACi) and hypomethylating agents yield synergistic effect in growth inhibition of malignant DLBCL cells. This effect has been demonstrated in in vitro analysis of cell viability, apoptosis induction, caspase system activation and histone acetylation in permanent and primary DLBCL lines utilizing the relative risk ration (RRR) calculations. Efficacy of decitabine and panobinostat (LBH589) in malignant cell growth inhibition has also been confirmed in in vivo human lymphoma xenograft model with a significant statistical difference (p < 0.05) in the mean tumour volume located on the flank of the animal in the group that received combination of decitabine and panobinostat compared to the single drug groups or the one that received vector only. Genome wide methylation analysis and gene expression profiling has been used to determine mechanistic and molecular basis for the synergistic effect of this combination. These analyses identified three potential tumour suppressors, TP73, MXD4 and CAV1, the synthesis of which was reactivated in cells treated with panobinostat and decitabine. Findings from this thesis are a part of a wider analysis in an effort to establish a pharmacology/biology systems model within B-cell neoplasm context. Practically, it will be a foundation for a clinical trial exploring efficacy of epigenetically targeted therapy

Role of epigenetically oriented therapy in the diffuse large B-cell lymphoma

Epigenetski orijentirana terapija potencijalno predstavlja novi način liječenja hematoloških novotvorina, uključujući i difuzni B-velikostanični limfom (DLBCL), pogotovo onih slučajeva koji su rezistentni na standardnu terapiju ili u bolesnika koji relapsiraju. U ovoj disertaciji pokazano je kako inhibitori histonske deacetilaze (HDACi) i hipometilirajuće tvari polučuju sinergistički učinak u zaustavljanju rasta malignih DLBCL stanica. Ovaj učinak je dokazan in vitro u analizi stanične vijabilnosti, indukcije apoptoze, aktivacije kaspaznog sustava te acetilacije histona računajući omjer relativnog rizika (RRR) u trajnim i primarnim limfomskim linijama. Učinkovitost panobinostata (LBH589) i decitabina u zaustavljanju rasta malignih stanica je potvrđena i u in vivo modelu ljudskog limfomskog ksenografta uz statistički značajnu razliku (p < 0.05) u prosječnom volumenu tumora na boku životinje u grupama koje su primile kombinaciju panobinostata i decitabina u odnosu na grupe životinja tretirane lijekom pojedinačno ili kontrolu koja je primila samo vektor. Mehanističko i molekularno objašnjenje sinergističkom učinku ove kombinacije istraženo je analizom metilacije genoma i promjene genske ekspresije tehnologijom mikropostroja kojima su identificirana tri potencijalna tumor supresora, TP73, MXD4 i CAV1, čija je aktivnost reaktivirana u limfomskim stanicama koje su tretirane panobinostatom i decitabinom. Spoznaje iz ove disertacije uključene su u širu analizu kako bi se stvorio model farmakologije i biologije sustava za B-stanične novotvorine, a praktično se primijenile u kliničkom istraživanju s epigenetski usmjerenom terapijom.Epigenetically targeted therapy potentially represents a novel way of treating hematologic malignancies, including diffuse large B-cell lymphoma (DLBCL), especially those cases resistant to standard therapeutic options and in patients with relapsed disease. As shown in this thesis, histone deacetylase inhibitors (HDACi) and hypomethylating agents yield synergistic effect in growth inhibition of malignant DLBCL cells. This effect has been demonstrated in in vitro analysis of cell viability, apoptosis induction, caspase system activation and histone acetylation in permanent and primary DLBCL lines utilizing the relative risk ration (RRR) calculations. Efficacy of decitabine and panobinostat (LBH589) in malignant cell growth inhibition has also been confirmed in in vivo human lymphoma xenograft model with a significant statistical difference (p < 0.05) in the mean tumour volume located on the flank of the animal in the group that received combination of decitabine and panobinostat compared to the single drug groups or the one that received vector only. Genome wide methylation analysis and gene expression profiling has been used to determine mechanistic and molecular basis for the synergistic effect of this combination. These analyses identified three potential tumour suppressors, TP73, MXD4 and CAV1, the synthesis of which was reactivated in cells treated with panobinostat and decitabine. Findings from this thesis are a part of a wider analysis in an effort to establish a pharmacology/biology systems model within B-cell neoplasm context. Practically, it will be a foundation for a clinical trial exploring efficacy of epigenetically targeted therapy

Role of epigenetically oriented therapy in the diffuse large B-cell lymphoma

Epigenetski orijentirana terapija potencijalno predstavlja novi način liječenja hematoloških novotvorina, uključujući i difuzni B-velikostanični limfom (DLBCL), pogotovo onih slučajeva koji su rezistentni na standardnu terapiju ili u bolesnika koji relapsiraju. U ovoj disertaciji pokazano je kako inhibitori histonske deacetilaze (HDACi) i hipometilirajuće tvari polučuju sinergistički učinak u zaustavljanju rasta malignih DLBCL stanica. Ovaj učinak je dokazan in vitro u analizi stanične vijabilnosti, indukcije apoptoze, aktivacije kaspaznog sustava te acetilacije histona računajući omjer relativnog rizika (RRR) u trajnim i primarnim limfomskim linijama. Učinkovitost panobinostata (LBH589) i decitabina u zaustavljanju rasta malignih stanica je potvrđena i u in vivo modelu ljudskog limfomskog ksenografta uz statistički značajnu razliku (p < 0.05) u prosječnom volumenu tumora na boku životinje u grupama koje su primile kombinaciju panobinostata i decitabina u odnosu na grupe životinja tretirane lijekom pojedinačno ili kontrolu koja je primila samo vektor. Mehanističko i molekularno objašnjenje sinergističkom učinku ove kombinacije istraženo je analizom metilacije genoma i promjene genske ekspresije tehnologijom mikropostroja kojima su identificirana tri potencijalna tumor supresora, TP73, MXD4 i CAV1, čija je aktivnost reaktivirana u limfomskim stanicama koje su tretirane panobinostatom i decitabinom. Spoznaje iz ove disertacije uključene su u širu analizu kako bi se stvorio model farmakologije i biologije sustava za B-stanične novotvorine, a praktično se primijenile u kliničkom istraživanju s epigenetski usmjerenom terapijom.Epigenetically targeted therapy potentially represents a novel way of treating hematologic malignancies, including diffuse large B-cell lymphoma (DLBCL), especially those cases resistant to standard therapeutic options and in patients with relapsed disease. As shown in this thesis, histone deacetylase inhibitors (HDACi) and hypomethylating agents yield synergistic effect in growth inhibition of malignant DLBCL cells. This effect has been demonstrated in in vitro analysis of cell viability, apoptosis induction, caspase system activation and histone acetylation in permanent and primary DLBCL lines utilizing the relative risk ration (RRR) calculations. Efficacy of decitabine and panobinostat (LBH589) in malignant cell growth inhibition has also been confirmed in in vivo human lymphoma xenograft model with a significant statistical difference (p < 0.05) in the mean tumour volume located on the flank of the animal in the group that received combination of decitabine and panobinostat compared to the single drug groups or the one that received vector only. Genome wide methylation analysis and gene expression profiling has been used to determine mechanistic and molecular basis for the synergistic effect of this combination. These analyses identified three potential tumour suppressors, TP73, MXD4 and CAV1, the synthesis of which was reactivated in cells treated with panobinostat and decitabine. Findings from this thesis are a part of a wider analysis in an effort to establish a pharmacology/biology systems model within B-cell neoplasm context. Practically, it will be a foundation for a clinical trial exploring efficacy of epigenetically targeted therapy

Uloga epigenetski usmjerene terapije u liječenju difuznog B-velikostaničnog limfoma [Role of epigenetically oriented therapy in the diffuse large B-cell lymphoma]

Epigenetically targeted therapy potentially represents a novel way of treating hematologic malignancies, including diffuse large B-cell lymphoma (DLBCL), especially those cases resistant to standard therapeutic options and in patients with relapsed disease. As shown in this thesis, histone deacetylase inhibitors (HDACi) and hypomethylating agents yield synergistic effect in growth inhibition of malignant DLBCL cells. This effect has been demonstrated in in vitro analysis of cell viability, apoptosis induction, caspase system activation and histone acetylation in permanent and primary DLBCL lines utilizing the relative risk ration (RRR) calculations. Efficacy of decitabine and panobinostat (LBH589) in malignant cell growth inhibition has also been confirmed in in vivo human lymphoma xenograft model with a significant statistical difference (p < 0.05) in the mean tumour volume located on the flank of the animal in the group that received combination of decitabine and panobinostat compared to the single drug groups or the one that received vector only. Genome wide methylation analysis and gene expression profiling has been used to determine mechanistic and molecular basis for the synergistic effect of this combination. These analyses identified three potential tumour suppressors, TP73, MXD4 and CAV1, the synthesis of which was reactivated in cells treated with panobinostat and decitabine. Findings from this thesis are a part of a wider analysis in an effort to establish a pharmacology/biology systems model within B-cell neoplasm context. Practically, it will be a foundation for a clinical trial exploring efficacy of epigenetically targeted therapy

The combination of hypomethylating agents and histone deacetylase inhibitors produce marked synergy in preclinical models of T-cell lymphoma

T-cell lymphomas (TCL) are aggressive lymphomas usually treated with CHOP (cyclophsophamide, doxorubicin, vincristine, prednisolone)-like regimens upfront. Recent data suggest that TCL are driven by epigenetic defects, potentially rendering them sensitive to epigenetic therapies. We explored the therapeutic merits of a combined epigenetic platform using histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMT) in in\ua0vitro and in\ua0vivo models of TCL. The 50% inhibitory concentration (IC50 ) values revealed romidepsin was the most potent HDACI, with an IC50 in the low nanomolar range. The combination with a hypomethylating agent produced synergy across all cell lines, which was confirmed in cytotoxicity and apoptosis assays. An in\ua0vivo xenograft study demonstrated inhibition of tumour growth in the combination cohort compared to the single agent. Gene expression array and global methylation profiling revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. Most of the effects induced by the single agent treatment were maintained in the combination group. In total, 944 unique genes were modulated by the combination treatment, supporting the hypothesis of molecular synergism. These data suggest combinations of hypomethylating agents and HDACIs are synergistic in models of TCL, which is supported at the molecular level