Exquisite regulation of PI3K/AKT/mTORC1 signaling is essential for homeostatic control of cell growth, proliferation, and survival. Aberrant activation of this signaling network is an early driver of ...many sporadic human cancers. Paradoxically, sustained hyperactivation of the PI3K/AKT/mTORC1 pathway in nontransformed cells results in cellular senescence, which is a tumor-suppressive mechanism that must be overcome to promote malignant transformation. While oncogene-induced senescence (OIS) driven by excessive RAS/ERK signaling has been well studied, little is known about the mechanisms underpinning the AKT-induced senescence (AIS) response. Here, we utilize a combination of transcriptome and metabolic profiling to identify key signatures required to maintain AIS. We also employ a whole protein-coding genome RNAi screen for AIS escape, validating a subset of novel mediators and demonstrating their preferential specificity for AIS as compared with OIS. As proof of concept of the potential to exploit the AIS network, we show that neurofibromin 1 (NF1) is upregulated during AIS and its ability to suppress RAS/ERK signaling facilitates AIS maintenance. Furthermore, depletion of NF1 enhances transformation of p53-mutant epithelial cells expressing activated AKT, while its overexpression blocks transformation by inducing a senescent-like phenotype. Together, our findings reveal novel mechanistic insights into the control of AIS and identify putative senescence regulators that can potentially be targeted, with implications for new therapeutic options to treat PI3K/AKT/mTORC1-driven cancers.
The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of ...these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.
The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited ...by the lack of animal models that reproduce plaque instability observed in humans.
Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis.
On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E-deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques.
The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.
In recent years fatty acid metabolism has gained greater attention in haematologic cancers such as acute myeloid leukaemia. The oxidation of fatty acids provides fuel in the form of ATP and NADH, ...while fatty acid synthesis provides building blocks for cellular structures. Here, we will discuss how leukaemic cells differ from healthy cells in their increased reliance on fatty acid metabolism. In order to understand how these changes are achieved, we describe the main pathways regulating fatty acid metabolism at the transcriptional level and highlight the limited knowledge about related epigenetic mechanisms. We explore these mechanisms in the context of leukaemia and consider the relevance of the bone marrow microenvironment in disease management. Finally, we discuss efforts to interfere with fatty acid metabolism as a therapeutic strategy along with the use of metabolic parameters as biomarkers.
Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused ...by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18.
Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) ...transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.
•Inhibition of RNA Pol I by CX-5461 treats aggressive AML and outperforms standard chemotherapy regimens.•CX-5461 induces p53-dependent apoptosis, p53-independent cell-cycle defects and differentiation, and reduces LICs.
Mutations in genes encoding chromatin regulators are early events contributing to developing asymptomatic clonal hematopoiesis of indeterminate potential and its frequent progression to myeloid ...diseases with increasing severity. We focus on the subset of myeloid diseases encompassing myelodysplastic syndromes and their transformation to secondary acute myeloid leukemia. We introduce the major concepts of chromatin regulation that provide the basis of epigenetic regulation. In greater detail, we discuss those chromatin regulators that are frequently mutated in myelodysplastic syndromes. We discuss their role in the epigenetic regulation of normal hematopoiesis and the consequence of their mutation. Finally, we provide an update on the drugs interfering with chromatin regulation approved or in development for myelodysplastic syndromes and acute myeloid leukemia.
RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by ...CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.
Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers ...and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.
Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to ...function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity).
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