scholarly journals Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation

2019 ◽  
Author(s):  
Abdalla Akef ◽  
Kathy McGraw ◽  
Steven D. Cappell ◽  
Daniel R. Larson
Keyword(s):  
Myeloid Leukemia ◽  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Common Mutation ◽  
Splice Site Selection ◽  
Downstream Effector ◽  
Processing Defects ◽  
Heterodimeric Complex ◽  
Global Increase ◽  
Missense Substitution

AbstractU2AF1 forms a heterodimeric complex with U2AF2 that is primarily responsible for 3’ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome and Acute Myeloid Leukemia, and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). However, the U2AF heterodimer also has a non-canonical function as a translational regulator. Here, we report that the U2AF1 S34F mutation results in specific mis-regulation of the translation initiation and ribosome biogenesis machinery, with the potential for widespread translational changes. The net result is a global increase in mRNA translation at the single cell level. Among the translationally upregulated targets of U2AF1-S34F are Nucleophosmin1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of wt/S34F cells and causes rRNA processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1 and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation which recapitulates translational mis-regulation in myeloid disease.

scholarly journals Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000920
Author(s):  
Abdalla Akef ◽  
Kathy McGraw ◽  
Steven D. Cappell ◽  
Daniel R. Larson
Keyword(s):  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Common Mutation ◽  
Small Nuclear Rna ◽  
Splice Site Selection ◽  
Downstream Effector ◽  
Heterodimeric Complex ◽  
Auxiliary Factor ◽  
Mutant Cells ◽  
Missense Substitution

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

scholarly journals New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Zaira Ianniello ◽  
Melissa Sorci ◽  
Lavinia Ceci Ginistrelli ◽  
Alessia Iaiza ◽  
Marcella Marchioni ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Ribosome Biogenesis ◽  
Kinase Inhibitors ◽  
Myeloproliferative Neoplasm ◽  
Mrna Translation ◽  
Translation Efficiency ◽  
Expression Of Genes ◽  
First Choice

AbstractChronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

Integrin Linked Kinase Is Expressed in Acute Myeloid Leukemia Blasts and Its Inhibition Is Cytotoxic to Leukemic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 154-154
Author(s):  
Andrew Muranyi ◽  
Shokat Dedhar ◽  
Donna E. Hogge
Keyword(s):  
Acute Myeloid Leukemia ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Akt Phosphorylation ◽  
Downstream Effector ◽  
Integrin Linked Kinase ◽  
Dose Dependent Decrease ◽  
Gsk 3Β ◽  
Acute Myeloid

Abstract Constitutive activation of the phosphatidylinositol-3-kinase (PI-3K) pathway is frequent in acute myeloid leukemia (AML) blasts and down-regulation of this pathway results in apoptotic death of these cells from many patient samples. Integrin linked kinase (ILK) is stimulated by activated PI-3K and ILK, in turn, activates AKT, a key downstream effector of the PI-3K pathway. The expression and activity of ILK are increased in a range of solid tumors. Small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumour growth, invasion and angiogenesis. We investigated the possible role of ILK in AML blast and colony forming cell (AML-CFC) survival. Using Western blotting, ILK protein was detected in 30 of 35 primary AML blast samples although the levels seen were variable. ILK kinase activity correlated strongly with ILK protein expression as quantitated by densitometry (r = 0.91) in 6 samples where both were studied. Activation of the PI-3K pathway, as measured by detection of AKT phosphorylation on serine 473 (ser473), was also found in 27 of 30 samples expressing ILK protein. QLT0267 is a potent second generation small molecule inhibitor (from QLT Inc., Vancouver, Canada) which selectively inhibits the kinase activity of ILK but not a variety of other kinases. Treatment of AML blasts with QLT0267 resulted in a time and dose dependent decrease in p-AKT ser473 expression as well as downstream targets of AKT (p-S6, p-GSK-3β). 27 AML and 5 normal bone marrow (NBM) samples were incubated for 48h with QLT0267 or 10 μM of the PI-3K inhibitor, LY294002, and then plated in CFC assay. There was a direct correlation between the % AML-CFC kill seen with LY204002 and QLT0267 (r= 0.70 and 0.60 comparing % kill with LY294002 and QLT0267 at 3 and 10 μM, respectively). The IC50 of QLT0267 was ≤ 3μM for 6 of 23 AML samples and 0 of 5 NBM samples (range % kill of AML and normal CFC after treatment with 3μM; 0 – 86% and 0 – 23%, respectively). The IC90 for this inhibitor was ≤ 10μM for 9 of 27 AML and 0 of 5 NBMs (range % kill 6 – 99 and 30 – 68, respectively, for AML and normal CFC treated with 10μM QLT0267). Interestingly, AML-CFC from 4 AML samples in which ILK protein could not be detected were resistant to killing with QLT0267. Thus, ILK is expressed in a large proportion of AML samples. Inhibition of ILK kinase is cytotoxic to leukemic progenitors suggesting that this molecule is important for the survival of these cells. Approximately one third of AML samples tested were more susceptible to killing by ILK inhibition than NBM cells suggesting that selective targeting of malignant rather than normal hematopoietic cells may be achieved in some cases. Furthermore, it may be possible to predict which AML samples will be sensitive to inhibitors such as QLT0267 by measuring expression of the target protein.

Specific MiRNA Silencing by Lentivirus Mediated Antagomir Expression in Chronic Myeloid Leukemia (CML) Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1017-1017
Author(s):  
Michaela Scherr ◽  
Letizia Venturini ◽  
Karin Battmer ◽  
Michael Schaller-Schoenitz ◽  
Daniel Schaefer ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mrna Translation ◽  
K562 Cells ◽  
Mature Mirnas ◽  
Dependent Manner ◽  
Aberrant Expression ◽  
Over Expression ◽  
Micro Rnas

Abstract Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′ untranslated region of corresponding mRNAs. Concomitant recruitment of specific multi-protein complexes results either in inhibition of mRNA translation or mRNA degradation. miRNAs are processed in a regulated multi-step process from primary transcripts into mature miRNAs by cellular components which are also at least partially involved in the process of RNA interference (RNAi). Aberrant expression of specific miRNAs has recently been described in human lymphoma and leukemia. In particular, BCR-ABL and c-MYC dependent over-expression of the polycistronic and oncogenic miR-17-92 cluster (encoding miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been described in chronic myeloid leukemia (CML) cell lines, primary CD34+ cells from CML patients (Venturini et al. 2007), and in lung cancer. In BCR-ABL positive K562 cells, miR-17-92 encoded miRNAs repress luciferase activity in miRNA-specific reporter assays. In addition, lentivirus-mediated over-expression of miR-17-92 increases both cell proliferation and sensitivity to imatinib induced cell death. To analyse the function of individual miRNAs of the miR-17-92 polycistron, we generated lentivirus-based strategies to induce stable miRNA-specific loss- and gain-of function phenotypes for miR-18a, miR-19b, and miR-20a, respectively. Over-expression of miRNAs embedded within miR-30-derived sequences from an internal SFFV-LTR promoter allows isolation of K562 cells with increased miRNA expression. In contrast, expression of complementary oligonucleotides (antagomirs) from a H1 promoter located in the lentiviral 3′LTR can induce stable hypomorphic miRNA-phenotypes. In lentivirally transduced K562 cells, individual silencing of miR-18a, miR-19b, and miR-20a by the corresponding antagomirs (ant-miR-18a, ant-miR-19b, ant-miR-20a) specifically relieves miRNA-mediated reporter gene repression. Correspondingly, inhibition of miRNA-function correlates to reduced ‘miRNA’-amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals specific induction of cell proliferation by miR-18a but strong inhibition by miR-20a in K562 cells, respectively. In contrast, anti-miR-18a, but not anti-miR-19b, anti-miR-20a, or control antagomirs inhibits proliferation of K562 cells. These data demonstrate individual and complementary functions of miR-17-92 encoded miRNAs in CML and identify potential targets for specific therapeutic intervention on the miRNA level.

The Musashi 2 mRNA Expression in the Course of CML.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2552-2552
Author(s):  
Sylvie Nadvornikova ◽  
Marketa Zackova ◽  
Tereza Lopotova ◽  
Hana Klamova ◽  
Jana Moravcova
Keyword(s):  
Myeloid Leukemia ◽  
Rna Binding ◽  
Blast Crisis ◽  
Mrna Level ◽  
Mrna Translation ◽  
Transcript Level ◽  
Response To Therapy ◽  
Expression Level ◽  
Molecular Response ◽  
Clinical Prognosis

Abstract Abstract 2552 The Musashi (MSI) gene family members, MSI1 and MSI2, represent an evolutionarily conserved family of RNA-binding proteins that regulate mRNA translation through binding in their N-termini. High levels of MSI2 protein are associated with increased cell proliferation, decreased cell maturation, more aggressive hematologic malignancy diseases and worse clinical prognosis. Recently obtained data pointed to MSI2 playing an important role in acute myeloid leukemia (AML) and in deadly blast crisis of chronic myeloid leukemia (CML) (Ito et al. 2010 Nature 5; 466). In this study we screened the level of MSI2 mRNA in 49 patients in different phases of CML and with different response to therapy – 18 patients at diagnosis (DG), 5 in major molecular response (MMR), 4 in complete molecular response (CMR), 2 after bone marrow transplantation (BMT), 10 in hematology relaps (HR), 6 in accelerrated phase (AP), and 4 in blast crisis (BC), and in 6 healthy donors. The level of MSI2 mRNA was quantified by real-time reverse-transcriptase-polymerase chain reaction using in-house designed specific primers and TaqMan probe and normalized to B2M endogenous control. Expression ratios were calculated by ΔΔCt method, and the differences between groups were statistically evaluated using Mann Whitney test. We detected MSI2 expression in all samples. The median expression of mRNA MSI2 in patients at DG was 1,43 (0,33–3,28), in MMR 0,52 (0,20–0,62), in CMR 0,37 (0,30–0,63), after BMT 1,28 (1,02–1,54), in HR 0,41 (0,16–0,58), in AP 3,78 (1,94–13,69), in BC 15,17 (2,61–28,15). MSI2 expression was statistically up-regulated in patients in advanced phases of CML (AP, BC) when compared with patients in CP (P<0.0001). The difference between patients in DG and remaining patients in CP was also statistically significant (P= 0,0006). No correlation of MSI2 expression level in DG patients with their responsiveness to treatment, BCR-ABL transcript level or survival was found. No significant differences were observed among groups of patients in MMR, CMR, HR, and after BMT. In addition, in order to check whether MSI2 expression level can serve as a marker of CML progression we also retrospectively screened kinetics of MSI2 transcript in 5 CML patients monitored on average 27 months (18–48). During this period, 3 patients developed HR, 1 patient AP and 1 BC. In BC patient the MSI2 transcript level increased with progression of CML in accordance with the increase of leucocytes and BCR-ABL transcript level. In 1 patient with a rising AP BCR-ABL levels remained constant compared to sevenfold increase of the MSI2 transcript level. On the other hand in HR patients we detected a constant or even decreasing level of MSI2 transcript regardless of the increase of leucocytes and BCR-ABL. In summary, our results confirm the association of high MSI2 mRNA level with advanced phases of CML and indicate that increase of MSI2 mRNA level may serve as a valuable marker of advanced phases of CML. In particular for CML patients with constantly high level of BCR-ABL mRNA the monitoring of MSI2 level can be important tool for early recognition of CML progression. Potential contributions of MSI2 to leukemic pathogenesis and its regulation in CML progression remain unknown. Grant support: NT/12392-4 IGA MZ-CR. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells

2017 ◽  
Vol 114 (30) ◽  
pp. E6117-E6126 ◽  
Author(s):  
Thomas C. J. Tan ◽  
John Knight ◽  
Thomas Sbarrato ◽  
Kate Dudek ◽  
Anne E. Willis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Ribosome Biogenesis ◽  
Cell Activation ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cell Receptor ◽  
Tcr Signaling

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

scholarly journals The DHX33 RNA Helicase Promotes mRNA Translation Initiation

2015 ◽  
Vol 35 (17) ◽  
pp. 2918-2931 ◽  
Author(s):  
Yandong Zhang ◽  
Jin You ◽  
Xingshun Wang ◽  
Jason Weber
Keyword(s):  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Rna Helicase ◽  
Rna Helicases ◽  
80S Ribosome ◽  
Protein Levels ◽  
Cell Growth And Proliferation ◽  
Rna Complexes

DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.

scholarly journals Gab2 deficiency prevents Flt3-ITD driven acute myeloid leukemia in vivo

Leukemia ◽  
2021 ◽  
Author(s):  
Corinna Spohr ◽  
Teresa Poggio ◽  
Geoffroy Andrieux ◽  
Katharina Schönberger ◽  
Nina Cabezas-Wallscheid ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinases ◽  
Myeloid Leukemia ◽  
Downstream Effector ◽  
Flt3 Inhibitor ◽  
Genes Encoding ◽  
Deficient Mice ◽  
Acute Myeloid

AbstractInternal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.

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