scholarly journals Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis

2019 ◽  
Author(s):  
E. P. Kusnadi ◽  
A. S. Trigos ◽  
C. Cullinane ◽  
D. L. Goode ◽  
O. Larsson ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Single Agent ◽  
Acquired Resistance ◽  
Cellular Metabolism ◽  
Mrna Translation ◽  
Pol I ◽  
Polymerase I

AbstractElevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA Polymerase I (Pol I) transcription, revealed single agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here we show that this improved efficacy is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this co-treatment is driven by translational re-wiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies identify the molecular mechanisms underpinning the response of blood cancers to selective ribosome biogenesis inhibitors and identify metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

scholarly journals The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eva Torreira ◽  
Jaime Alegrio Louro ◽  
Irene Pazos ◽  
Noelia González-Polo ◽  
David Gil-Carton ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Mutational Analysis ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rdna Transcription ◽  
Pol I ◽  
Polymerase I

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

scholarly journals Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy

2021 ◽  
Author(s):  
Xin Peng ◽  
Shaolu Zhang ◽  
Wenhui Jiao ◽  
Zhenxing Zhong ◽  
Yuqi Yang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Single Agent ◽  
Antitumor Efficacy ◽  
Antitumor Effects ◽  
Autophagy Inhibitor

Abstract Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

scholarly journals SLC36A1-mTORC1 signaling drives acquired resistance to CDK4/6 inhibitors

2019 ◽  
Vol 5 (9) ◽  
pp. eaax6352 ◽  
Author(s):  
Akihiro Yoshida ◽  
Yiwen Bu ◽  
Shuo Qie ◽  
John Wrangle ◽  
E. Ramsay Camp ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fragile X ◽  
Acquired Resistance ◽  
Mammalian Target Of Rapamycin ◽  
Cyclin Dependent Kinase ◽  
Solute Carrier Family ◽  
Potential Therapeutic Target ◽  
Mtorc1 Signaling ◽  
Solute Carrier

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

scholarly journals Structure and function of ribosomal RNA gene chromatin

2008 ◽  
Vol 36 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Joanna L. Birch ◽  
Joost C.B.M. Zomerdijk
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pol I ◽  
And Function ◽  
Polymerase I ◽  
Cell Growth And Proliferation ◽  
Cellular Control

Transcription of the major ribosomal RNAs by Pol I (RNA polymerase I) is a key determinant of ribosome biogenesis, driving cell growth and proliferation in eukaryotes. Hundreds of copies of rRNA genes are present in each cell, and there is evidence that the cellular control of Pol I transcription involves adjustments to the number of rRNA genes actively engaged in transcription, as well as to the rate of transcription from each active gene. Chromatin structure is inextricably linked to rRNA gene activity, and the present review highlights recent advances in this area.

scholarly journals RNA Polymerase I-Promoted HIS4Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (2) ◽  
pp. 665-675 ◽  
Author(s):  
Hsiu-Jung Lo ◽  
Han-Kuei Huang ◽  
Thomas F. Donahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Mutant Form ◽  
Wild Type ◽  
Pol I ◽  
Chromosome Iii ◽  
Polymerase I

ABSTRACT The HIS4 gene in Saccharomyces cerevisiaewas put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. Therhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-typeHIS4 mRNA. Its 3′ end maps to the HIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l, is bicistronic. It encodes the HIS4coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3′ end of rhis4l maps to the predicted 3′ end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5′ end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5′-3′ exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-typeHIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1Δ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5′ cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

scholarly journals Tumour initiating cells and IGF/FGF signalling contribute to sorafenib resistance in hepatocellular carcinoma

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 530-540 ◽  
Author(s):  
Victoria Tovar ◽  
Helena Cornella ◽  
Agrin Moeini ◽  
Samuel Vidal ◽  
Yujin Hoshida ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Growth Factor ◽  
Molecular Mechanisms ◽  
Acquired Resistance ◽  
Gene Signature ◽  
Signalling Pathways ◽  
Fgf Signalling

ObjectiveSorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance.DesignHCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts.ResultsSorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC).ConclusionsAcquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.

scholarly journals CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5056
Author(s):  
Robert Cornelison ◽  
Kuntal Biswas ◽  
Danielle C. Llaneza ◽  
Alexandra R. Harris ◽  
Nisha G. Sosale ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Time Frame ◽  
Interferon Response ◽  
Type I ◽  
Pol I ◽  
Downstream Effects ◽  
Immune Pathway ◽  
Polymerase I

Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of ‘STimulator of Interferon Genes’ (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP–AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS–STING–TBK1–IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.

scholarly journals Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Cinzia Lanzi ◽  
Enrica Favini ◽  
Laura Dal Bo ◽  
Monica Tortoreto ◽  
Noemi Arrighetti ◽  
...  
Keyword(s):  
Synovial Sarcoma ◽  
Cell Response ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Combined Treatment ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Xenograft Model

Abstract Background Synovial sarcoma (SS) is an aggressive soft tissue tumor with limited therapeutic options in advanced stage. SS18-SSX fusion oncogenes, which are the hallmarks of SS, cause epigenetic rewiring involving histone deacetylases (HDACs). Promising preclinical studies supporting HDAC targeting for SS treatment were not reflected in clinical trials with HDAC inhibitor (HDACi) monotherapies. We investigated pathways implicated in SS cell response to HDACi to identify vulnerabilities exploitable in combination treatments and improve the therapeutic efficacy of HDACi-based regimens. Methods Antiproliferative and proapoptotic effects of the HDACi SAHA and FK228 were examined in SS cell lines in parallel with biochemical and molecular analyses to bring out cytoprotective pathways. Treatments combining HDACi with drugs targeting HDACi-activated prosurvival pathways were tested in functional assays in vitro and in a SS orthotopic xenograft model. Molecular mechanisms underlying synergisms were investigated in SS cells through pharmacological and gene silencing approaches and validated by qRT-PCR and Western blotting. Results SS cell response to HDACi was consistently characterized by activation of a cytoprotective and auto-sustaining axis involving ERKs, EGR1, and the β-endoglycosidase heparanase, a well recognized pleiotropic player in tumorigenesis and disease progression. HDAC inhibition was shown to upregulate heparanase by inducing expression of the positive regulator EGR1 and by hampering negative regulation by p53 through its acetylation. Interception of HDACi-induced ERK-EGR1-heparanase pathway by cell co-treatment with a MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/roneparstat) enhanced antiproliferative and pro-apoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and nuclear heparanase levels. The combination of SAHA with SST0001 prevented the upregulation of ERK-EGR1-heparanase induced by the HDACi and promoted caspase-dependent cell death. In vivo, the combined treatment with SAHA and SST0001 potentiated the antitumor efficacy against the CME-1 orthotopic SS model as compared to single agent administration. Conclusions The present study provides preclinical rationale and mechanistic insights into drug combinatory strategies based on the use of ERK pathway and heparanase inhibitors to improve the efficacy of HDACi-based antitumor therapies in SS. The involvement of classes of agents already clinically available, or under clinical evaluation, indicates the transferability potential of the proposed approaches.

scholarly journals The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Lucas Tafur ◽  
Yashar Sadian ◽  
Jonas Hanske ◽  
Rene Wetzel ◽  
Felix Weis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Reversible Binding ◽  
Cryo Electron Microscopy ◽  
Nucleotide Analog ◽  
Pol I ◽  
Polymerase I ◽  
Insight Into

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.

scholarly journals Peptides Targeting the Interaction Between Erb1 and Ytm1 Ribosome Assembly Factors

2021 ◽  
Vol 8 ◽  
Author(s):  
Lidia Orea-Ordóñez ◽  
Susana Masiá ◽  
Jerónimo Bravo
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
Ribosome Biogenesis ◽  
Acquired Resistance ◽  
Biological Action ◽  
Pol I ◽  
Assembly Factors ◽  
Multistep Process ◽  
Cellular Partner

Ribosome biogenesis is an emerging therapeutic target. It has been proposed that cancer cells are addicted to ribosome production which is therefore considered a druggable pathway in cancer therapy. Cancer cells have been shown to be more sensitive to inhibition of the ribosome production than healthy cells. Initial attempts of inhibiting ribosome biogenesis have been focused on the inhibition of transcription by targeting RNA Pol I. Despite being a promising field of research, several limitations have been identified during the development of RNA Pol I inhibitors, like the lack of specificity or acquired resistance. Ribosome biogenesis is a multistep process and additional points of intervention, downstream the very initial stage, could be investigated. Eukaryotic ribosome maturation involves the participation of more than 200 essential assembly factors that will not be part of the final mature ribosome and frequently require protein–protein interactions to exert their biological action. Using mutagenesis, we have previously shown that alteration of the complex interface between assembly factors impairs proper ribosome maturation in yeast. As a first step toward the developing of ribosome biogenesis inhibitory tools, we have used our previously solved crystal structure of the Chaetomium thermophilum complex between the assembly factors Erb1 and Ytm1 to perform a structure-guided selection of interference peptides. The peptides have been assayed in vitro for their ability to bind their cellular partner using biophysical techniques.

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