RESUMO
Acute myeloid leukemia (AML) is one of the leading leukemic malignancies in adults. The heterogeneity of the disease makes the diagnosis and treatment extremely difficult. With the advent of next-generation sequencing (NGS) technologies, exploration at the molecular level for the identification of biomarkers and drug targets has been the focus for the researchers to come up with novel therapies for better prognosis and survival outcomes of AML patients. However, the huge amount of data from NGS platforms requires a comprehensive AML platform to streamline literature mining efforts and save time. To facilitate this, we developed AMLdb, an interactive multi-omics platform that allows users to query, visualize, retrieve, and analyse AML related multi-omics data. AMLdb contains 86 datasets for gene expression profiles, 15 datasets for methylation profiles, CRISPR-Cas9 knockout screens of 26 AML cell lines, sensitivity of 26 AML cell lines to 288 drugs, mutations in 41 unique genes in 23 AML cell lines, and information on 41 experimentally validated biomarkers. In this study, we have reported five genes, i.e. CBFB, ENO1, IMPDH2, SEPHS2, and MYH9 identified via our analysis using AMLdb. ENO1 is uniquely identified gene which requires further investigation as a novel potential target while other reported genes have been previously confirmed as targets through experimental studies. Top of form we believe that these findings utilizing AMLdb can make it an invaluable resource to accelerate the development of effective therapies for AML and assisting the research community in advancing their understanding of AML pathogenesis. AMLdb is freely available at https://project.iith.ac.in/cgntlab/amldb.
RESUMO
Chromosomal translocations are considered as one of the major causes of lymphoid cancers. RAG complex, which is responsible for V(D)J recombination, can also cleave non-B DNA structures and cryptic RSSs in the genome leading to chromosomal translocations. The mechanism and factors regulating the illegitimate function of RAGs resulting in oncogenesis are largely unknown. Upon in silico analysis of 3760 chromosomal translocations from lymphoid cancer patients, we find that 93% of the translocation breakpoints possess adjacent cryptic nonamers (RAG binding sequences), of which 77% had CpGs in proximity. As a proof of principle, we show that RAGs can efficiently bind to cryptic nonamers present at multiple fragile regions and cleave at adjacent mismatches generated to mimic the deamination of CpGs. ChIP studies reveal that RAGs can indeed recognize these fragile sites on a chromatin context inside the cell. Finally, we show that AID, the cytidine deaminase, plays a significant role during the generation of mismatches at CpGs and reconstitute the process of RAG-dependent generation of DNA breaks both in vitro and inside the cells. Thus, we propose a novel mechanism for generation of chromosomal translocation, where RAGs bind to the cryptic nonamer sequences and direct cleavage at adjacent mismatch generated due to deamination of meCpGs or cytosines.
Assuntos
Neoplasias , Translocação Genética , Humanos , Cromatina , Citidina Desaminase/genética , DNA/genética , Proteínas de Homeodomínio/metabolismo , Neoplasias/genética , Translocação Genética/genética , Ilhas de CpGRESUMO
Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
Assuntos
Proteínas de Homeodomínio/química , Ácidos Nucleicos Heteroduplexes/química , Motivos de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Recombinação V(D)JRESUMO
BACKGROUND: Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. RESULTS: We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. CONCLUSION: This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Células-Tronco Embrionárias , Feminino , Humanos , Masculino , CamundongosRESUMO
Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.
Assuntos
Instabilidade Cromossômica , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência Conservada , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Linfócitos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , FosforilaçãoRESUMO
Integrase inhibitors are a class of antiretroviral drugs used for the treatment of AIDS that target HIV integrase, an enzyme responsible for integration of viral cDNA into host genome. RAG1, a critical enzyme involved in V(D)J recombination exhibits structural similarity to HIV integrase. We find that two integrase inhibitors, Raltegravir and Elvitegravir, interfered with the physiological functions of RAGs such as binding, cleavage and hairpin formation at the recombination signal sequence (RSS), though the effect of Raltegravir was limited. Circular dichroism studies demonstrated a distinct change in the secondary structure of RAG1 central domain (RAG1 shares DDE motif amino acids with integrases), and when incubated with Elvitegravir, an equilibrium dissociation constant (Kd) of 32.53±2.9 µM was determined by Biolayer interferometry, leading to inhibition of its binding to DNA. Besides, using extrachromosomal assays, we show that Elvitegravir inhibited both coding and signal joint formation in pre-B cells. Importantly, treatment with Elvitegravir resulted in significant reduction of mature B lymphocytes in 70% of mice studied. Thus, our study suggests a potential risk associated with the use of Elvitegravir as an antiretroviral drug, considering the evolutionary and structural similarities between HIV integrase and RAGs.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , Proteínas de Homeodomínio/genética , Células Precursoras de Linfócitos B/efeitos dos fármacos , Quinolonas/farmacologia , Recombinação V(D)J/efeitos dos fármacos , Animais , Sítios de Ligação , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Linhagem Celular , Linhagem Celular Transformada , Células HEK293 , Integrase de HIV/química , Integrase de HIV/genética , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/química , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/virologia , Cultura Primária de Células , Ligação Proteica , Quinolonas/química , Raltegravir Potássico/química , Raltegravir Potássico/farmacologiaRESUMO
Pyrazole moiety represents an important category of heterocyclic compound in pharmaceutical and medicinal chemistry. The novel 1-aryl-3, 5-bis (het) aryl pyrazole derivatives were synthesized with complementary regioselectivity. The chemical structures were confirmed by IR, 1H NMR, 13C NMR, and mass spectral analysis. The chemical entities were screened in various cancer cell lines to assess their cell viability activity. Results showed that the compound 3-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl) pyridine (5d) possessed maximum cytotoxic effect against breast cancer and leukemic cells. The cytotoxicity was confirmed by live-dead cell assay and cell cycle analysis. Mitochondrial membrane potential, Annexin V-FITC staining, DNA fragmentation, Hoechst staining, and western blot assays revealed the ability of compound 5d to induce cell death by activating apoptosis in cancer cells. Thus, the present study demonstrates that compound 5d could be an attractive chemical entity for the development of small molecule inhibitors for treatment of leukemia and breast cancer.
Assuntos
Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Citotoxinas , Leucemia/tratamento farmacológico , Pirazóis , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Citotoxinas/síntese química , Citotoxinas/química , Citotoxinas/farmacologia , Feminino , Humanos , Células K562 , Leucemia/metabolismo , Células MCF-7 , Espectrometria de Massas , Camundongos , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologiaRESUMO
Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy.
Assuntos
Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Quercetina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ácido Elágico/administração & dosagem , Ácido Elágico/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Quercetina/administração & dosagem , Análise de Sobrevida , Resultado do TratamentoRESUMO
The RAG (recombination-activating gene) complex is responsible for the generation of antigen receptor diversity by acting as a sequence-specific nuclease. Recent studies have shown that it also acts as a structure-specific nuclease. However, little is known about the factors regulating this activity at the genomic level. We show in the present study that the proximity of a V(D)J nonamer to heteroduplex DNA significantly increases RAG cleavage and binding efficiencies at physiological concentrations of MgCl(2). The position of the nonamer with respect to heteroduplex DNA was important, but not orientation. A spacer length of 18 bp between the nonamer and mismatch was optimal for RAG-mediated DNA cleavage. Mutations to the sequence of the nonamer and deletion of the nonamer-binding domain of RAG1 reinforced the role of the nonamer in the enhancement in RAG cleavage. Interestingly, partial mutation of the nonamer did not significantly reduce RAG cleavage on heteroduplex DNA, suggesting that even cryptic nonamers were sufficient to enhance RAG cleavage. More importantly, we show that the fragile region involved in chromosomal translocations associated with BCL2 (B-cell lymphoma 2) can be cleaved by RAGs following a nonamer-dependent mechanism. Hence our results from the present study suggest that a non-B DNA can replace the heptamer of RSS (recombination signal sequence) when present adjacent to nonamers, explaining the generation of certain chromosomal translocations in lymphoid malignancies.