Large Scale Screening for DNA Damage-Induced Transcription Factors as Potential Targets for Treatment of CLL with p53 Apoptotic Defect.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3439-3439
Author(s):  
Victoria Weston ◽  
Paul Moss ◽  
A. Malcolm R Taylor ◽  
Tatjana Stankovic
Keyword(s):  
Dna Damage ◽  
Transcription Factors ◽  
Dna Binding ◽  
Aryl Hydrocarbon Receptor ◽  
P53 Pathway ◽  
Binding Motifs ◽  
Aryl Hydrocarbon ◽  
Dna Damaging Agents ◽  
Dna Binding Motifs ◽  
The Impact

Abstract Abstract 3439 Poster Board III-327 Chronic lymphocytic leukaemia (CLL) is a malignancy with a variable clinical course in which a proportion of patients exhibits rapid clinical progression despite treatment. One of the major causes of treatment resistance is alterations in the ATM/p53 pathway imposed by mutations in either the ATM or TP53 genes. Consequently, there is an urgent need to devise novel therapeutic approaches that will be able to counteract the p53 apoptotic defect in these tumours. We have previously shown that DNA damage induces a complex ATM-dependent network of pro-survival and pro-apoptotic transcriptional responses (both p53-dependent and -independent) and that the balance between these responses determines CLL cellular death. Therefore, it is plausible to expect that manipulation of ATM-dependent transcription to either reduce pro-survival or increase pro-apoptotic signals can sensitise ATM and TP53 mutant CLL tumours to DNA damaging agents. Individual transcription factors (TFs) that govern ATM-dependent transcription are largely unknown. In this study we aimed to identify those factors by employing a DNA/Protein Transcription Factor ComboArray (Panomics/Affymetrix) which includes 345 DNA binding motifs for a range of transcription factors, DNA binding proteins and response elements. We compared the ability of nuclear cell extracts from 3 combined ATM wildtype primary CLL samples and 3 combined ATM mutant primary CLL samples to bind to biotin-labelled DNA binding motifs prior to irradiation (IR)-induced DNA damage, 2h and 6h post-IR. Following hybridisation of nuclear protein-bound biotin-labelled probes to the array and HRP visualisation, we identified 49 binding motifs (several of which were detected more than once through alternative sequences) which, in response to DNA damage, exhibited reduced binding in ATM mutant compared to the ATM wildtype CLL nuclear extracts. The most prominent differentially bound DNA binding motifs included those for GATA1 and 2, Transcriptional enhancer factor 1 (TEF1), c-Rel, Aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator binding element (AhR/Arnt), forkhead box I1 (HFH-3), Slow/Cardiac Troponin C (cTnC/CEF-2), E2A immunoglobulin enhancer binding factors (E12/E47), Pax-4, Wilms tumour 1 (WT1), antioxidant recognition element (ARE) and interferon-a stimulated response element (ISRE). We validated differential binding of individual TFs by electro-mobility shift analysis (EMSA) and selected six that were positively corroborated in an independent cohort of primary ATM mutant and ATM wildtype CLL tumour cells. We subsequently investigated the impact of altering the activity of the identified ATM-dependent TFs on the sensitivity of ATM mutant CLL tumours to DNA damage. Among the selected TFs, as a proof of principle, ARE demonstrated both ATM-dependent binding by EMSA as well as the capacity to modulate the DNA damage response in CLL cells: pharmacological activation of this TF by Dimethyl fumarate (DMF) sensitised ATM mutant cells to IR-induced DNA damage. In summary, we have identified a number of ATM-regulated transcription factors that could be directly or indirectly targeted to increase the sensitivity of CLL cells with a defective ATM/p53 pathway to DNA damaging agents. We also suggest that the DNA damage-dependent TF screen represents a feasible approach to identify novel molecular targets that may sensitise other subtypes of treatment-resistant CLL tumours. Disclosures No relevant conflicts of interest to declare.

ATR Inhibition Exacerbates Replication Stress in TP53 or ATM Deficient CLL Cells and Enhances Sensitivity to Chemotherapy and Targeted Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3340-3340 ◽  
Author(s):  
Marwan Kwok ◽  
Nicholas Davies ◽  
Angelo Agathanggelou ◽  
Edward Smith ◽  
Eva Petermann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Clonal Evolution ◽  
Replication Stress ◽  
Disease Relapse ◽  
P53 Pathway ◽  
Dna Damaging Agents ◽  
Signaling Inhibitors ◽  
Xenograft Models ◽  
The Impact

Abstract DNA damage response (DDR) defects, particularly TP53 or biallelic ATM aberrations, are associated with chemoresistance in chronic lymphocytic leukemia (CLL). Chemoimmunotherapy or B-cell receptor signaling inhibitors alone may not be sufficient to overcome adverse prognosis or provide durable response in TP53 or biallelic ATM inactivated CLL. In particular, genomic instability resulting from impaired DDR facilitates rapid clonal evolution leading to treatment refractoriness or disease relapse. Development of therapeutic approaches specifically targeting DDR defects is therefore necessary for effective long-term control of DDR-defective CLL. We previously demonstrated selective cytotoxicity of the ATR inhibitor AZD6738 towards TP53 or ATM null CLL cells, and validated this in CLL xenograft models for biallelic TP53 or ATM loss. Here, we provide mechanistic insight into the synthetically lethal interactions between ATR pathway inhibition and TP53 or ATM loss in CLL, and offer experimental evidence supporting the use of ATR inhibition in combination with conventional chemotherapies and other targeted therapies in CLL. To determine the mechanism of ATR inhibition in CLL, we first investigated its effect on DDR-intact cycling primary cells. We observed compensatory activation of the ATM/p53 pathway in AZD6738-treated cells in response to hydroxyurea, providing evidence for crosstalk between ATR and ATM/p53 pathways. Next, we explored the cellular consequence of ATR inhibition in CLL cells with DDR loss. To determine the impact of ATR inhibition on DNA replication, we performed DNA fiber analysis, which revealed significantly increased replication fork stalling and firing of replication origins upon AZD6738 treatment in ATM/p53-defective CLL. To investigate the effect of ATR inhibition on DNA damage, we measured γH2AX and 53BP1 foci formation, markers of DNA double-strand breaks. Significant induction of γH2AX and 53BP1 foci was seen in ATM/p53-defective CLL cells upon AZD6738 treatment, suggesting that ATR inhibition exacerbates replication stress in ATM/p53 defective cells by imposing requirement for DDR through the ATM/p53 pathway. On the contrary, 53BP1 bodies, a marker of unreplicated DNA, appeared in ATM/p53 proficient but not in ATM/p53 deficient cells following AZD6738 exposure, indicating that cell cycle arrest in response to replication stress is ATM/p53 dependent. This was corroborated by cell cycle profiling and co-labeling experiments of γH2AX with phosphohistone H3 ser10, a marker of mitosis, showing that ATM/p53-defective CLL cells carrying unrepaired DNA damage continued to cycle. Thus, ATR inhibition results in accumulation of intolerable levels of DNA damage in DDR-defective CLL, leading to cell death by mitotic catastrophe, which we have confirmed both in vitro and in AZD6738-treated murine xenograft models. The benefit of ATR inhibition as a therapeutic strategy for DDR-defective CLL lies in its ability to alter the subclonal landscape in favor of less unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of disease relapse. We reasoned that this provides a strong rationale for addition of AZD6738 to existing therapeutic agents for the treatment of CLL with DDR defects. We demonstrated synergistic and additive effects of low-dose AZD6738 in ATM/p53-defective CLL cells with DNA damaging agents such as chlorambucil, fludarabine, bendamustine and cyclophosphamide, BCR-signaling inhibitors and the PARP inhibitor olaparib. We validated the AZD6738 plus chlorambucil combination in biallelic TP53 or ATM inactivated primary CLL xenografts, where combined AZD6738/chlorambucil treatment was superior to chlorambucil alone, as evidenced by significantly greater reduction in tumor load and percentage of CLL subclones with del(17p) or del(11q) in animals treated with combination regimen compared to single-agent chlorambucil. Similar investigations evaluating AZD6738 plus ibrutinib versus ibrutinib monotherapy in primary CLL xenografts are currently underway. We conclude that ATR inhibition is a suitable approach for targeting the loss of p53 function in aggressive CLL subclones and should be considered as a valuable addition to DNA damaging agents and current targeted treatments. Disclosures Off Label Use: ATR inhibitor AZD6738 targets TP53-null or ATM-null phenotype inducing synthetic lethality. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

scholarly journals RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Toro-Moreno ◽  
Kayla Sylvester ◽  
Tamanna Srivastava ◽  
Dora Posfai ◽  
Emily R. Derbyshire
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Redox Homeostasis ◽  
Early Time ◽  
Rna Seq ◽  
Binding Motifs ◽  
Liver Stage ◽  
Content Type ◽  
Dna Binding Motifs ◽  
Causative Agents

ABSTRACT The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive transcriptome sequencing (RNA-Seq) analysis throughout the Plasmodium berghei LS, covering as early as 2 h postinfection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite’s LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCE The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite’s biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provide insights into these early parasite fluxes that may facilitate establishment of infection, transformation, and replication in the liver.

scholarly journals Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes

2014 ◽  
Vol 42 (22) ◽  
pp. 13500-13512 ◽  
Author(s):  
Mario Pujato ◽  
Fabien Kieken ◽  
Amanda A. Skiles ◽  
Nikos Tapinos ◽  
Andras Fiser
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
3D Models ◽  
Binding Motifs ◽  
Dna Binding Motifs

Missense Mutations Located in Structural p53 DNA-Binding Motifs Are Associated With Extremely Poor Survival in Chronic Lymphocytic Leukemia

2011 ◽  
Vol 29 (19) ◽  
pp. 2703-2708 ◽  
Author(s):  
Martin Trbusek ◽  
Jana Smardova ◽  
Jitka Malcikova ◽  
Ludmila Sebejova ◽  
Petr Dobes ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Dna Binding ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Functional Assay ◽  
Missense Mutations ◽  
P53 Mutations ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
The Impact

Purpose There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. Patients and Methods We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. Results A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). Conclusion The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

scholarly journals ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors

2015 ◽  
Vol 25 (6) ◽  
pp. 825-835 ◽  
Author(s):  
Stephan R. Starick ◽  
Jonas Ibn-Salem ◽  
Marcel Jurk ◽  
Céline Hernandez ◽  
Michael I. Love ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Insight Into

scholarly journals A Rice Promoter Protein Binding Microarray for Cis-Acting Elements for Rice Transcription Factors

2020 ◽  
Author(s):  
JOUNG SUG KIM ◽  
SongHwa Chae ◽  
Kyong Mi Jun ◽  
Gang-Seob Lee ◽  
Jong-Seong Jeon ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Protein Binding ◽  
Target Genes ◽  
Specific Protein ◽  
Transcriptional Level ◽  
Binding Motifs ◽  
Putative Target ◽  
Protein Binding Microarray ◽  
Dna Binding Motifs

Abstract BackgroundTranscription factors (TFs) regulate the expression of genes at the transcriptional level by binding a specific DNA sequence. Thus, predicting the DNA-binding motifs of TFs is one of the most important areas for the functional analysis of TFs in the postgenomic era. Although many methods have been developed for this challenge, there are still many TFs with unknown DNA-binding motifs.FindingsIn this paper, we designed an rice (Oryza sativa)-specific protein binding microarray (RPBM), and its probes are 40 bp long with 20 bp of overlap; there are 49 probes spanning the 1 kb promoter region before the translation start site of each gene. To confirm the efficiency of RPBM technology, we selected two TFs, OsWOX13 and OsSMF1. We identified the ATTGATTG DNA-binding sequence and 635 putative target genes of OsWOX13. OsSMF1 bound to GCTGACTCA and GGATGCC sequences and bound especially strongly to CCACGTCA. A total of 932 putative target genes were identified for OsSMF1.ConclusionsRPBM can be applicable in the analysis of DNA-binding motifs for TFs where binding is evaluated in extended natural promoter regions. The analysis can also be applicable to TFs that have single or multiple binding motifs. The technology might even be expanded for application to TFs that are heterodimers or form higher-order complexes.

scholarly journals Comparative Analysis of the IclR-Family of Bacterial Transcription Factors and Their DNA-Binding Motifs: Structure, Positioning, Co-Evolution, Regulon Content

2021 ◽  
Vol 12 ◽  
Author(s):  
Inna A. Suvorova ◽  
Mikhail S. Gelfand
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Dna Interaction ◽  
Orthologous Genes ◽  
Binding Motifs ◽  
Bacterial Transcription ◽  
Alternating Direction ◽  
Dna Binding Motifs ◽  
General Protein

The IclR-family is a large group of transcription factors (TFs) regulating various biological processes in diverse bacteria. Using comparative genomics techniques, we have identified binding motifs of IclR-family TFs, reconstructed regulons and analyzed their content, finding co-occurrences between the regulated COGs (clusters of orthologous genes), useful for future functional characterizations of TFs and their regulated genes. We describe two main types of IclR-family motifs, similar in sequence but different in the arrangement of the half-sites (boxes), with GKTYCRYW3–4RYGRAMC and TGRAACAN1–2TGTTYCA consensuses, and also predict that TFs in 32 orthologous groups have binding sites comprised of three boxes with alternating direction, which implies two possible alternative modes of dimerization of TFs. We identified trends in site positioning relative to the translational gene start, and show that TFs in 94 orthologous groups bind tandem sites with 18–22 nucleotides between their centers. We predict protein–DNA contacts via the correlation analysis of nucleotides in binding sites and amino acids of the DNA-binding domain of TFs, and show that the majority of interacting positions and predicted contacts are similar for both types of motifs and conform well both to available experimental data and to general protein–DNA interaction trends.

scholarly journals RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

2019 ◽  
Author(s):  
Maria Toro-Moreno ◽  
Kayla Sylvester ◽  
Tamanna Srivastava ◽  
Dora Posfai ◽  
Emily R. Derbyshire
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Redox Homeostasis ◽  
Early Time ◽  
Clinical Malaria ◽  
Rna Seq ◽  
Binding Motifs ◽  
Liver Stage ◽  
Dna Binding Motifs ◽  
Causative Agents

ABSTRACTThe apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood when compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive RNA-seq analysis throughout the Plasmodium berghei LS, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi, and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with co-expression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding presents potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite’s LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites.IMPORTANCEThe LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite’s biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provides insights into these early parasite fluxes that may facilitate establishment of infection, transformation and replication in the liver.

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