A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs

A phase I study of a dual PI3-kinase/mTOR inhibitor BEZ235 in adult patients with relapsed or refractory acute leukemia

Background Combined inhibition of phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) complexes may be an efficient treatment for acute leukemia. The primary objective of this phase I single center open label study was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of the dual pan-class I PI3K and mTOR inhibitor BEZ235 in patients with advanced leukemia. Methods Herein patients > 18 years of age who had relapsed or showed refractory leukemia were treated with BEZ235 (orally at 300–400 mg BID (cohort − 1/1)) to assess safety, tolerability, preliminary efficacy and pharmacokinetic (PK). Adverse events data and serious adverse events were analyzed and haematological and clinical biochemistry toxicities were assessed from laboratory test parameters. Response was assessed for the first time at the end of cycle 1 (day 29) and after every subsequent cycle. Pharmacokinetic and pharmacodynamic analyses of BEZ235 were also included (BEZ235 plasma levels, phosphorylation of AKT, S6 and 4EBP1). On statistics this trial is a multiple ascending dose study in which a following variant of the 3 + 3 rule (“Rolling Six”), a minimum of 6 and a maximum of 12 patients was recruited for the dose escalation and another 5 were planned for the expansion phase. Results Twenty-four patients with ALL (n = 11) or AML (n = 12) or CML-BP (n = 1) were enrolled. All patients had failed one (n = 5) or more lines of therapy (n = 5) and 14 patients were in refractory / refractory relapse. No formal MTD was defined, stomatitis and gastrointestinal toxicity at 400 mg BID dose was considered incompatible with prolonged treatment. The RP2D of BEZ235 was defined as 300 mg BID. Four of 24 patients showed clinical benefit. Twenty-two of 24 patients discontinued because of progression, (median time to progression 27 days (4d-112d). There was no association between PK parameters and efficacy or tolerability. Conclusions Combined inhibition of PI3K and mTOR inhibits a clinically meaningful driver pathway in a small subset of patients with ALL, with no benefit in patients with AML

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1 that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs

Die Akkumulation von mutiertem p53 in humanen Krebszellen

Der Tumorsuppressor p53 ist in über 50% aller humaner, maligner Tumore mutiert. Dies beinhalted meist Punktmutationen einzelner Aminosäuren, die einen Verlust der Tumorsuppressor Funktion nach sich ziehen. Des Weiteren konnte gezeigt werden, dass mutiertes p53 onkogene Eigenschaften, wie erhöhte Invasivität und Chemoresistenz, mit sich bringt. Mutiertes p53 Protein akkumuliert speziell in den Zellen fortgeschrittener Tumore, jedoch nicht in den umliegenden Geweben, auch wenn diese ebenfalls entsprechende Punktmutationen in dem Gen aufweisen. Diese Beobachtung legt nahe, dass Veränderungen während der Tumorigenese diese Akkumulation bewirken. Im Rahmen dieser Arbeit haben wir gezeigt, dass der Level an mutiertem p53 zusätzlich ansteigt, sobald die Zellen einer Behandlung mit manchen, aber nicht allen, Chemotherapeutika ausgesetzt werden. Während die Anthrazykline Doxorubicin, Daunorubicin und Epirubicin zu einer Akkumulation von mutiertem p53 führen, haben wir beobachtet, dass das eng verwandte Molekül Idarubicin keine Auswirkungen zeigt. Unseren Untersuchungen zur Folge geschieht die Regulation von mutiertem p53 auf verschiedenen Ebenen: Erstens, führt die Behandlung mit den Topoisomerase II Inhibitoren Daunorubicin, Doxorubicin, Epirubicin, Idarubicin und Etoposide zu einer DNA Schadensantwort, die unter anderem die Aktivierung von E2F1 und seinem Zielgen TAp73 zur Folge hat. Unsere Daten sprechen weiterhin dafür, dass diese genotoxische Behandlung zu einer E2F1-vermittelten Aktivierung von mutierter p53 prä-mRNA Synthese führt, sowohl direkt, also auch durch TAp73. Außerdem zeigen wir, dass der Transkriptionsfaktor E2F1 selbst mit der Promoter DNA von TP53 assoziiert. Zweitens, unter den genannten Chemotherapeutika, die p53 Transkription induzieren, haben wir 2 Vertreter gefunden die zusätzlich ein natürliches Antisense Transkript, WRAP53, induzieren. Weitergehend haben wir beobachtet, dass die Induktion von WRAP53 mit einer verhinderten p53 mRNA Reifung einhergeht. Daher stellen wir die Hypothese auf, dass die Expression des Antisense Transkripts mit der Stabilität der prä-mRNA oder ihres Kernexports interferiert. Drittens, die Akkumulation von mutiertem p53 Protein, die im Rahmen der Tumorigenese beobachtet wird, ist hauptsächlich auf einen Regulationsdefekt auf Proteinebene zurück zu führen. Wir haben mit Hilfe von Einzelzell basierter Fluoreszenzmikroskopie einen Hochdurchsatz siRNA Screen durchgeführt und in diesem Zusammenhang die ribosomalen S6 Kinasen als Regulatoren von mutierter p53 Expression identifiziert. Wir glauben, dass unsere Beobachtungen bei der Verschreibung von Chemotherapeutika berücksichtigt werden sollten, nachdem wir zeigen konnten, dass einige Toposiomerase II inhibitoren die Expression von mutiertem p53 steigern und somit unerwünschte Nebenwirkungen erzielen können

Abstract B54: Identification of the PI3K-mTOR pathway as regulator of mutant p53 turnover in human cancer cells

Abstract The tumor suppressor p53 plays a crucial role in our cells to induce cell cycle arrest or apoptosis in response to different kinds of genotoxic stresses. In 50% of all solid tumors, p53 is mutated by a single point mutation within the DNA-binding domain and thereby loses its activity as a tumor suppressor. However, at the same time, mutant p53 actively contributes to chemoresistance and tumor progression through a gain of function (Dittmer et al., 1993). It was previously shown that mutant p53 highly accumulates in tumors of mice with targeted germ line mutations of the p53 gene (knock-in), whereas normal cells of these mice express it at low levels (Lang et al., 2004; Terzian et al., 2008). This argues that the deregulation of tumor-specific signaling cascades leads to an accumulation of mutant p53 and subsequently to tumor progression. Our study aims at identifying these signaling pathways that contribute to the regulation of mutant p53 expression, and to describe their mechanisms. We performed quantitative analysis of mutant p53 protein levels in a cancer cell line after the individual knock-down of all known human kinases using automated microscopy (high content cell screening). The bladder carcinoma derived cell line 5637 (carrying the p53 R280T mutation) was transfected with three different synthetic siRNAs targeting each known kinase or kinase regulator in a 96-well format, and p53 was detected by automated immunofluorescence microscopy. In each of the resulting 2400 cell populations the p53 expression levels were determined for at least 500 individual cells. Alterations in the distribution of p53 staining intensities were assessed for each kinase relative to non-targeting control siRNAs. The results of the kinases, that lead to the strongest up- or down-regulations of mutant p53 in the screen, were confirmed in different cell lines (U251 and U373, both derived from glioma, with the p53 hot spot mutation R273H), by immunofluorescence and immunoblot analysis. We identified a group of kinases currently known to act in the PI3-kinase → mTOR pathway as potential regulators of mutant p53. The knock-down of these kinases resulted in a decrease of mutant p53 expression. Studying the mechanism of mutant p53 regulation, we performed quantitative real-time PCR after siRNA transfection and found no significant impairment of the p53 transcription. On the contrary, posttranslational modifications (phosphorylation and acetylation) of mutant p53 were decreased after knock-down of the identified kinases, even more strongly than the overall levels of the protein. These observations support the idea that the degradation of mutant p53 is decreased in cancer cells through alterations of PI3K-mTOR pathway associated kinases, resulting in p53 accumulation and oncogenic gain of function. Citation Information: Cancer Res 2009;69(23 Suppl):B54.</jats:p

A phase I study of a dual PI3-Kinase/mTOR inhibitor BEZ235 in adult patients with relapsed or refractory acute leukemia

Abstract Background: Combined inhibition of phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) complexes may be an efficient treatment for acute leukemia. The primary objective of this phase I single center open label study was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of the dual pan-class I PI3K and mTOR inhibitor BEZ235 in patients with advanced leukemia.Methods: Herein patients &gt; 18 years of age who had relapsed or showed refractory leukemia were treated with BEZ235 (orally at 300-400 mg BID (cohort -1/1)) to assess safety, tolerability, preliminary efficacy and pharmacokinetic (PK). Adverse events data and serious adverse events were analyzed and haematological and clinical biochemistry toxicities were assessed from laboratory test parameters. Response was assessed for the first time at the end of cycle 1 (day 29) and after every subsequent cycle. Pharmacokinetic and pharmacodynamic analyses of BEZ235 were also included (BEZ235 plasma levels, phosphorylation of AKT, S6 and 4EBP1). On statistics this trial is a multiple ascending dose study in which a following variant of the 3+3 rule (“Rolling Six”), a minimum of 6 and a maximum of 12 patients was recruited for the dose escalation and another 5 were planned for the expansion phase.Results: Twenty-four patients with ALL (n=11) or AML (n=12) or CML-BP (n=1) were enrolled. All patients had failed one (n=5) or more lines of therapy (n=5) and 14 patients were in refractory / refractory relapse. No formal MTD was defined, stomatitis and gastrointestinal toxicity at 400 mg BID dose was considered incompatible with prolonged treatment. The RP2D of BEZ235 was defined as 300 mg BID. Four of 24 patients showed clinical benefit. Twenty-two of 24 patients discontinued because of progression, (median time to progression 27 days (4d-112d). There was no association between PK parameters and efficacy or tolerability.Conclusions: Combined inhibition of PI3K and mTOR inhibits a clinically meaningful driver pathway in a small subset of patients with ALL, with no benefit in patients with AML.Trial registration: ClinicalTrials.gov, identifier NCT01756118. retrospectively registered 19th December 2012, https://clinicaltrials.gov/ct2/show/NCT01756118.</jats:p

A phase I study of a dual PI3-Kinase/mTOR inhibitor BEZ235 in adult patients with relapsed or refractory acute leukemia

Abstract Background Combined inhibition of phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) complexes may be an efficient treatment for acute leukemia. The primary objective of this phase I single centre open lable study was to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the dual pan-class I PI3K and mTOR inhibitor BEZ235 in patients with advanced leukemia.Methods Herein patients &gt; 18 years of age who had relapsed or showed refractory leukemia were treated with BEZ235 (orally at 300-400 mg BID (cohort -1/1)) to assess safety, tolerability, preliminary efficacy and pharmakokinetic (PK). Adverse events data and serious adverse events were analyzed and haematological and clinical biochemistry toxicities were assessed from laboratory test parameters. Response was assessed for the first time at the end of cycle 1 (day 29) and after every subsequent cycle. Pharmacokinetic and pharmacodynamic analyses of BEZ235 were also included (BEZ235 plasma levels, phosphorylation of AKT, S6 and 4EBP1). On statistics this trial is a multiple ascending dose study in which a following variant of the 3+3 rule (“Rolling Six”), a minimum of 6 and a maximum of 12 patients was recruited for the dose escalation and another 5 were planned for the expansion phase. Results Twenty-four patients with ALL (n=11) or AML (n=12) or CML-BP (n=1) were enrolled. All patients had failed one (n=5) or more lines of therapy (n=5) and 14 patients were in refractory / refractory relapse. No formal MTD was defined, stomatitis and gastrointestinal toxicity at 400 mg BID dose was considered incompatible with prolonged treatment. The RP2D of BEZ235 was defined as 300 mg BID. Four of 24 patients showed clinical benefit. Twenty-two of 24 patients discontinued because of progression, (median time to progression 27 days (4d-112d). There was no association between PK parameters and efficacy or tolerability. Conclusions Combined inhibition of PI3K and mTOR inhibits a clinically meaningful driver pathway in a small subset of patients with ALL, with no benefit in patients with AML. Trial registration ClinicalTrials.gov, identifier NCT01756118. registered 19th Decembre 2012, https://clinicaltrials.gov/ct2/show/NCT01756118</jats:p