Präklinische und klinische Charakterisierung von DNA-Methyltransferase Inhibitoren als Wirkstoffe für die Tumortherapie

Veränderte Methylierungsmuster spielen eine wichtige Rolle bei der Entstehung von Krebserkrankungen. Insbesondere die Hypermethylierung von Tumorsuppressorgenen ist ein häufiges Ereignis, das die Stilllegung von Genen verursacht. Es konnte gezeigt werden, dass die Inhibition der DNA-Methyltransferasen (DNMTs) mit DNMT-Inhibitoren zur Reaktivierung von epigenetisch stillgelegten Genen führen kann. Die beiden DNA-Methyltranferase Inhibitoren 5-Azacytidin und Decitabin haben bereits zu einer klinischen Verbesserung in der Behandlung von Myelodysplastischen Syndromen (MDS) geführt. Beide Substanzen wurden von der FDA (Food and Drug Administration) für die Behandlung dieser Erkrankungen zugelassen. Nichtsdestotrotz sind die demethylierenden Eigenschaften dieser Substanzen bisher nicht gut charakterisiert worden. Daher war das Ziel meiner Arbeit die demethylierenden und wachstumsinhibierenden Eigenschaften von DNMT Inhibitoren genauer zu charakterisieren. Für diesen Zweck wurde ihre Wirkung in therapiebezogenen präklinischen Modellsystemen untersucht. Außerdem wurden klinische Proben aus MDS-Patienten analysiert, die mit 5-Azacytidin behandelt wurden. Ich konnte in Zellkulturmodellen zeigen, dass eine Behandlung mit 5-Azacytidin oder mit Decitabin, im Vergleich mit anderen DNMT-Inhibitoren die stärkste Inhibition der DNA-Methylierung zeigte. Überdies war auch die wachstumsinhibierende Wirkung dieser Substanzen am stärksten ausgeprägt. In weiteren Experimenten wurde die Rolle von Transporterproteinen für die inhibitorischen Wirkungen der Azanukleoside untersucht. Die Ergebnisse aus diesen Versuchen ergaben, dass die erleichterte Aufnahme von 5-Azacytidin und Decitabin in einer erhöhten Sensitivität gegenüber den demethylierenden und wachstumsinhibierenden Wirkungen dieser DNMT Inhibitoren resultierte. Ein wichtiger Aspekt für die klinische Charakterisierung von DNMT Inhibitoren war die Analyse der Methylierungsveränderungen in MDS-Patienten. Die Methylierungsuntersuchung von Blut und Knochenmarksproben aus MDS-Patienten, die mit 5-Azazcytidin behandelt wurden, ergab erhebliche Unterschiede in der DNA-Demethylierung zwischen individuellen Patienten. In drei von sechs Patienten konnte eine signifikante und transiente genomweite DNA-Demethylierung nachgewiesen werden. Eine detaillierte Array-basierte Analyse ergab, dass viele hypermethylierte Einzelgene demethyliert werden konnten. In Übereinstimmung mit diesen Ergebnissen zeigten leukämische Krebszellen bemerkenswerte Unterschiede in der durch die Behandlung induzierten Verringerung der DNA-Methyltransferasen. Diese Verringerung korreliert jeweils mit den beobachteten Methylierungs-änderungen in den jeweiligen Zelllinien. Insgesamt konnte in dieser Arbeit ein wichtiger Nachweis der demethylierenden Aktivität von 5-Azacytidin in MDS Patienten erbracht werden. Zusätzlich konnte die Existenz von zuvor unbekannten Faktoren aufgedeckt werden, die die Wirkungen von DNMT Inhibitoren beeinflussen

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors

Antiproliferative Effects of DNA Methyltransferase 3B Depletion Are Not Associated with DNA Demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs

Efficient depletion of DNMT3B has no effect on DNA methylation.

<p>(A) DNMT3B mRNA levels were determined after siRNA knockdown with DNMT3B siRNA #4 by quantitative RT-PCR analysis. Expression values are means of triplicates and were calculated relative to Lamin B1 expression. Error bars represent standard errors. ***P<0.001 relative to control siRNA, determined by student's t-test prior to normalization. (B) Comparison of Infinium 27 k methylation profiles of HCT-116 cells transfected with DNMT3B siRNA #4 or control siRNA. (C) Efficient depletion of DNMT3B protein in stably shRNA-transduced HCT-116 cells. DNMT3B protein levels were determined by immunoblot analysis using ß-actin as a loading control. The double band presumably reflects the expression of two (or more) DNMT3B isoforms. (D) Comparison of Infinium 27 k methylation profiles between HCT-116 cells stably transduced with lentiviruses containing DNMT3B shRNA #1 and control cells transfected with a control shRNA. (E) Infinium 450 k methylation analysis comparing methylation profiles of HCT-116 cells transduced with DNMT3B-shRNA #1 and control cells transduced with a non-targeting control shRNA. (F) Heatmap based on the Infinium 450 k data, showing hypermethylated CIMP markers in HCT-116 cells stably transduced with shRNA #1 or #2 targeting DNMT3B or with a non-targeting control shRNA. AVB, Average Beta value.</p

The hypermethylation phenotype is maintained in DNMT3B knockout cells.

<p>(A) Heatmap based on Infinium 27 k data illustrating the methylation levels of 67 CIMP marker genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (B) Boxplot illustrating methylation levels of 67 CIMP marker genes. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles, respectively. (C) Heatmap based on Infinium 27 k data illustrating the methylation levels of 543 hypermethylated PRC2 target genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (D) Boxplot illustrating methylation levels of PRC2 target genes. AVB, Average Beta value.</p

The hypermethylation phenotype is reversed in HCT-116 DNMT1; DNMT3B double knockout cells.

<p>(A, B) Boxplots illustrating the methylation levels of CIMP (A) and PRC2 (B) marker genes in parental HCT-116 cells (HCT-116), HCT-116 cells lacking DNMT3B (3BKO), HCT-116 cells with a hypomorphic DNMT1 allele (1KO), and HCT-116 DNMT1; DNMT3B double knockout cells (DKO) on Infinium 27 k methylation arrays. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. ***P<0.001 relative to parental HCT116 cells, determined by a Wilcoxon rank sum test. (C) Model for the role of DNMT3B during cancer development and progression. In normal cells, CpG islands are generally unmethylated and the corresponding genes are actively transcribed. During cancer development, certain regions become methylated by de novo methyltransferases such as DNMT3A and DNMT3B. Once the hypermethylation phenotype is established, the methylation pattern is maintained by the maintenance methyltransferase DNMT1 and becomes independent of DNMT3 enzymes. Only a strong reduction of overall methyltransferase activity causes the demethylation of hypermethylated cancer genes and inhibits the re-establishment by de novo methyltransferases.</p

DNMT3B expression in human cancer tissue and colon cancer cell lines.

<p>(A) Array Northern analysis of DNMT3B mRNA expression on Affymetrix HGU133Plus2.0 arrays in tumor (T) tissue samples compared to normal (N) tissues of the same organ. n indicates the number of analyzed samples for the specified tissue type. Data are shown as mean±SD; *P<0.05 relative to normal tissue (N), **P<0.01 relative to normal tissue (N), ***P<0.001 relative to normal tissue (N), determined by student's t-test. (B) Quantitative real-time PCR analysis of DNMT3B mRNA expression in colon cancer cell lines and normal cells (HMEC-T53, WI-38). Expression values are means of triplicates and were calculated relative to GAPDH expression. Error bars represent standard errors. (C) Immunoblot of DNMT3B protein expression in lysates from various colon cancer cell lines and normal cells (HMEC-T53, WI-38). ß-actin was used as a loading control.</p

Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer

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Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed