Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction

Background: genome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications. Results: profiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus. Conclusions: DNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses

Identification of functional vulnerabilities in acute myeloid leukemia

Obwohl sich das Outcome bei Patienten mit akuter myeloischer Leukämie (AML) in den letzten Jahrzehnten verbessert hat, liegt die Gesamtüberlebensrate jedoch immer noch unter 50 % [1, 2], und es besteht nach wie vor ein essentieller Bedarf an der Entwicklung neuer therapeutischer Strategien. In diesem Projekt haben wir funktionelle Vulnerabilitäten in der AML identifiziert und anschließend das therapeutische Potenzial von Zielstrukturen untersucht, die an der Proteostase, der Zellpolarität sowie an der RNA-Prozessierung bei verschiedenen Signalwegen beteiligt sind. Dabei konnten wir zeigen, dass die genetische Inaktivierung des Zellpolaritätsregulators Scribble die Entwicklung der AML verzögert. Jedoch scheint die Deletion von Scribble auch die Proliferationsfähigkeit normaler hämatopoetischer Zellen zu beeinträchtigen, was ein Angehen von Scribble als therapeutisches Zielstruktur in der AML erschwert. Im Gegensatz dazu hemmt sowohl die Inaktivierung des pleiotropen DNA- und RNA-bindenden Proteins YBX1, welches zur Familie der Cold-shock Proteine zählt, als auch die Inaktivierung der katalytischen Untereinheit PSMB8/LMP7 des Immunoproteasom-Multiproteinkomplexes, der zum Ubiquitin-Proteasom- System (UPS) gehört, das Wachstum von Leukämie-Zellen, ohne dabei die Funktion normaler hämatopoetischer Stamm- und Vorläuferzellen zu beeinflussen. Diese Tatsache prädistiniert diese Zielstrukturen als potenzielle neue therapeutische Strategien, die bei der Behandlung von AML-Patienten Anwendung finden könnten. Die genetische Inaktivierung von YBX1 führt zu einer verringerten Proliferation und Koloniebildungsfähigkeit in Leukämie-Zellen, unabhängig von der onkogenen Treibermutation, und verzögerte die Entwicklung einer AML in vivo. Mit Hilfe eines konditionalen Ybx1 Knockout-Modells konnten wir nachweisen, dass Ybx1 auch für die Aufrechterhaltung der AML in vivo eine Rolle spielt. Unsere Untersuchungen zeigen, dass YBX1 onkogene Transkripte, wie z.B. MYC, an die Polysomen rekrutiert und somit deren Translation verstärkt. Die Inaktivierung von YBX1 führt zu einer Verdrängung dieser Transkripte von den Polysomen und resultiert in einem Rückgang der onkogenen Proteinexpression. Die genetische und pharmakologische Hemmung von PSMB8/LMP7 verringert selektiv die Proliferation und die Fähigkeit zur Koloniebildung in KMT2A (MLL)-transformierten leukämischen Zellen. Die in vivo Behandlung mit einem PSMB8/LMP7-Inhibitor verzögert die Krankheitsentwicklung in einem KMT2Atransformierten Leukämie-Mausmodell ebenso wie in einem „Patient-derived“ Xenograft-Modell (PDX). In weiteren Untersuchungen konnten wir den transkriptionellen Korepressor BASP1 als funktionellen Effektor des Immunoproteasoms identifizieren. Nach Hemmung von PSMB8/LMP7 kommt es zu einer Akkumulation von BASP1, welches dann an KMT2A-Zielgene bindet. Darüber hinaus führt die pharmakologische Hemmung von PSMB8/LMP7 zu einer verminderten Expression von KMT2A-Zielgenen sowie zu einer Anreicherung von Genen, die durch Inhibitoren der KMT2A-Komplexpartner DOT1L und MEN1 dereguliert wurden. Die Kombinationsbehandlung von KMT2A -transformierten AML-Zellen mit einem Immunoproteasom-Inhibitor und einem Menin-Inhibitor führt im Vergleich zu der jeweiligen Mono-Behandlung zu einer deutlich verringerten Proliferation in vitro und einer erhöhten Überlebensrate in vivo. Diese Daten weisen auf ein therapeutisches Potenzial einer Kombinationbehandlung von Immunoproteasom- und Menin-Inhibitoren hin.Although the outcome of patients with acute myeloid leukemia (AML) has improved in the past decades, the overall survival is below 50% [1, 2] and there is still an unmet need for the development of new therapeutic strategies. Here, we aimed to identify functional vulnerabilities in AML and investigated the therapeutic potential of target structures involved in proteostasis, cell polarity and RNA-binding molecular pathways. We determined that genetic deletion of the cell fate determinant and polarity regulator Scribble delays AML development, however, its deletion also seems to affect the proliferative capacity of normal hematopoietic cells, lowering its value as a therapeutic target. In contrast, inactivation of YBX1 (a pleiotropic protein with DNA/RNA binding capacity that excerpts post-transcriptional control on its targets) and PSMB8/LMP7 (a catalytic subunit of the immunoproteasome multiprotein complex that belongs to the ubiquitin-proteasome system (UPS)) inhibit leukemic cells without influencing normal hematopoietic stem and progenitor cell function, establishing these targets as potential novel therapeutic strategies against AML. Genetic deletion of YBX1 caused reduced proliferation and colony forming capacity in leukemic cells independent of the oncogenic driver mutation and delayed AML development in vivo. The role of Ybx1 in leukemia maintenance was investigated using a conditional knockout model, confirming the functional requirement of Ybx1 in AML maintenance. Mechanistically, YBX1 recruited oncogenic transcripts to polysomes, increasing their translation. Displacement of these transcripts from polysomes after YBX1 deletion decreased their protein expression. Genetic and pharmacologic inhibition of PSMB8/LMP7 decreased proliferation and colony forming capacity selectively in KMT2A (MLL)-rearranged leukemic cells. In vivo treatment with a PSMB8/LMP7 inhibitor delayed disease development in KMT2A-rearranged leukemic mice or patient derived xenografts (PDX). We identified the transcriptional corepressor BASP1 as a functional effector of the immunoproteasome. BASP1 was enriched after PSMB8/LMP7 inhibition and it was found binding to KMT2A-target genes. Moreover, pharmacologic inhibition of PSMB8/LMP7 led to decreased expression of bonafide KMT2A-fusion target genes and enrichment for genes deregulated by inhibitors of the KMT2A complex partners DOT1L and MEN1. This prompted us to investigate a potential synergism between MEN1 inhibition and immunoproteasome inhibition. Combination treatment in AML cells revealed decreased proliferation in vitro and increased survival in vivo as compared to the single treatments, demonstrating the therapeutic potential of combining immunoproteasome and MEN1 inhibitors

Immunoproteasome Function in Normal and Malignant Hematopoiesis

The ubiquitin–proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers

Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia

Abstract Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML