Role of DDX RNA helicases in cancer cells

Ester Cannizzaro ROLE OF DDX RNA HELICASES IN CANCER CELLS Abstract DEAD-BOX (DDX) RNA helicases are a large family of proteins characterized by the presence of a DEAD/H (Asp-Glu-Ala-Asp/His) motif. Their main function is to unwind double stranded RNA to promote downstream molecular events. They are involved in virtually all steps of RNA metabolism such as transcription, translation, RNA export and degradation, ribosome biogenesis and pre-mRNA splicing. The aim of my work was to investigate the functions of human DDX3X and DDX54 RNA helicases, particularly in in vitro cancer models. To provide insight into their molecular functions, I identified RNAs bound by DDX3X and DDX54 in breast cancer (MCF7) cells by performing iCLIP experiments. This generated two very distinct RNA binding profiles: DDX3X preferentially bound exonic regions of mRNAs encoding translational factors, whilst DDX54 preferentially bound non-coding RNAs and intronic regions of mRNAs encoding nuclear proteins. Further bioinformatic analysis identified a few discrete binding motifs within DDX3X target RNAs. One of these, within the human JUND transcript, was validated as a DDX3X binding site using electrophoretic mobility shift assays. Notably, the levels of proteins encoded by mRNAs bound by DDX3X were altered following knockdown of the helicase. These data highlight the importance of DDX3X in maintaining appropriate levels of certain proteins, which in turn may explain at least some of the changes in phenotype observed upon DDX3X knockdown. In this regard, I showed that knocking down DDX3X or DDX54 in MCF7 cells slowed cell proliferation by inducing a G1/S phase arrest. Furthermore, a CRISPR/Cas9 dropout screen in a leukaemia cell line (MLL-AF9) showed that both helicases are essential for growth of these cells. However, loss of DDX3X or DDX54 had little effect on proliferation of immortalized NIH3T3 cells indicating that their loss is not generally lethal. In subsequent CRISPR/Cas9 dropout screens in various other cancer cell lines, including some derived from solid tumours, DDX54 was found to be essential for growth of all cell lines. In contrast, DDX3X was required for proliferation of only a subset of these. Focusing on DDX3X, I identified that the integrity of the helicase's RNA binding domains is essential for growth and cell cycle progression of an acute myeloid leukaemia cell line (OCI-AML3). Overall, my findings shed mechanistic insight upon the role of DDX RNA helicases in cancer and identify DDX3X and DDX54 as potential targets for therapeutic intervention in certain cancers

DDX3X RNA helicase affects breast cancer cell cycle progression by regulating expression of KLF4.

DDX3X is a multifunctional RNA helicase with documented roles in different cancer types. Here, we demonstrate that DDX3X plays an oncogenic role in breast cancer cells by modulating the cell cycle. Depletion of DDX3X in MCF7 cells slows cell proliferation by inducing a G1 phase arrest. Notably, DDX3X inhibits expression of Kruppel-like factor 4 (KLF4), a transcription factor and cell cycle repressor. Moreover, DDX3X directly interacts with KLF4 mRNA and regulates its splicing. We show that DDX3X-mediated repression of KLF4 promotes expression of S-phase inducing genes in MCF7 breast cancer cells. These findings provide evidence for a novel function of DDX3X in regulating expression and downstream functions of KLF4, a master negative regulator of the cell cycle

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis

Stress Perfusion Cardiac Magnetic Resonance in Long-Standing Non-Infarcted Chronic Coronary Syndrome with Preserved Systolic Function

(1) Background: The impact of imaging-derived ischemia is still under debate and the role of stress perfusion cardiac magnetic resonance (spCMR) in non-high-risk patient still needs to be clarified. The aim of this study was to evaluate the impact of spCMR in a case series of stable long-standing chronic coronary syndrome (CCS) patients with ischemia and no other risk factor. (2) Methods: This is a historical prospective study including 35 patients with history of long-standing CCS who underwent coronary CT angiography (CCTA) and additional adenosine spCMR. Clinical and imaging findings were included in the analysis. Primary outcomes were HF (heart failure) and all major cardiac events (MACE) including death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, or resuscitated cardiac arrest. (3) Results: Mean follow-up was 3.7 years (IQR: from 1 to 6). Mean ejection fraction was 61 ± 8%. Twelve patients (31%) referred primary outcomes. Probability of experiencing primary outcomes based on symptoms was 62% and increased to 67% and 91% when multivessel disease and ischemia, respectively, were considered. Higher ischemic burden was predictive of disease progression (OR: 1.59, 95%CI: 1.18–2.14; p-value = 0.002). spCMR model resulted non inferior to the model comprising all variables (4) Conclusions: In vivo spCMR-modeling including perfusion and strain anomalies could represent a powerful tool in long-standing CCS, even when conventional imaging predictors are missing

Stress Perfusion Cardiac Magnetic Resonance in Long-Standing Non-Infarcted Chronic Coronary Syndrome with Preserved Systolic Function

(1) Background: The impact of imaging-derived ischemia is still under debate and the role of stress perfusion cardiac magnetic resonance (spCMR) in non-high-risk patient still needs to be clarified. The aim of this study was to evaluate the impact of spCMR in a case series of stable long-standing chronic coronary syndrome (CCS) patients with ischemia and no other risk factor. (2) Methods: This is a historical prospective study including 35 patients with history of long-standing CCS who underwent coronary CT angiography (CCTA) and additional adenosine spCMR. Clinical and imaging findings were included in the analysis. Primary outcomes were HF (heart failure) and all major cardiac events (MACE) including death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, or resuscitated cardiac arrest. (3) Results: Mean follow-up was 3.7 years (IQR: from 1 to 6). Mean ejection fraction was 61 ± 8%. Twelve patients (31%) referred primary outcomes. Probability of experiencing primary outcomes based on symptoms was 62% and increased to 67% and 91% when multivessel disease and ischemia, respectively, were considered. Higher ischemic burden was predictive of disease progression (OR: 1.59, 95%CI: 1.18–2.14; p-value = 0.002). spCMR model resulted non inferior to the model comprising all variables (4) Conclusions: In vivo spCMR-modeling including perfusion and strain anomalies could represent a powerful tool in long-standing CCS, even when conventional imaging predictors are missing

Uterine fibroid therapy using interventional radiology mini-invasive treatments: current perspective

Uterine fibroids are common benign tumors of unclear etiopathology that affect the female reproductive tract. They are responsible for considerable morbidity and deterioration of life quality, and may have a negative impact on the reproductive system as well. Besides surgery aided by uterus-saving techniques, several minimally invasive procedures are now available within the field of interventional radiology that represent a valid solution for women who desire pregnancy and relief from disease-specific symptomatology. The main advantages offered by these techniques are low grade of invasiveness and short times of hospitalization. The most diffuse techniques are uterine artery embolization (UAE) and magnetic resonance-guided high-intensity focused ultrasound (MRgFUS). UAE is an endovascular procedure whose goal is obtained by provoking ischemia of the uterine vessels. MRgFUS is a thermoablation procedure that selectively ablates the symptomatic fibroids. In this review study, both procedures will be described, including a description of technical details, indications, contraindications, complications, and outcomes

Heart Failure and Cardiomyopathies: CT and MR from Basics to Advanced Imaging

Since 1997, heart failure (HF) has been designated as a new epidemic. However, it is not easy to find a proper definition since different descriptors are used in clinical practice. Moreover, HF is not a single clinical entity, and there is a close relationship between HF and all cardiomyopathies (CMs). This leads us to also consider accuracy in the characterization of CMs, which is essential to define the therapeutic process of HF patients. This narrative review aims to describe the main mechanisms leading to HF in different CMs, as well as the current diagnostic and prognostic advantages deriving from advanced imaging in the cardiac field

The Protein Landscape of Chronic Lymphocytic Leukemia (CLL)

Many functional consequences of mutations on tumor phenotypes in chronic lymphocytic leukemia (CLL) are unknown. This may be in part due to a scarcity of information on the proteome of CLL. We profiled the proteome of 117 CLL patient samples with data-independent acquisition mass spectrometry (DIA-MS) and integrated the results with genomic, transcriptomic, ex vivo drug response and clinical outcome data. We found trisomy 12, IGHV mutational status, mutated SF3B1, trisomy 19, del(17)(p13), del(11)(q22.3), mutated DDX3X, and MED12 to influence protein expression (FDR < 5%). Trisomy 12 and IGHV status were the major determinants of protein expression variation in CLL as shown by principal component analysis (1055 and 542 differentially expressed proteins, FDR=5%). Gene set enrichment analyses of CLL with trisomy 12 implicated BCR/PI3K/AKT signaling as a tumor driver. These findings were supported by analyses of protein abundance buffering and protein complex formation, which identified limited protein abundance buffering and an upregulated protein complex involved in BCR, AKT, MAPK and PI3K signaling in trisomy 12 CLL. A survey of proteins associated with trisomy 12/IGHV-independent drug response linked STAT2 protein expression with response to kinase inhibitors including BTK and MEK inhibitors. STAT2 was upregulated in U-CLL, trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a non-redundant layer of information in tumor biology, and provides a protein expression reference map for CLL

Multi-omics reveals clinically relevant proliferative drive associated with mTOR-MYC-OXPHOS activity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex vivo drug response data from 217 patients with CLL. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n = 547 patients). We found that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL–PD was linked to the activation of mTOR–MYC–oxidative phosphorylation through transcriptomic, proteomic and single-cell resolution analysis. CLL–PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.ISSN:2662-134