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Histone methyltransferases (HMTs) are enzymes that regulate histone methylation and play an important role in controlling transcription by altering the chromatin structure. Aberrant activation of HMTs has been widely reported in certain types of neoplastic cells. Among them, G9a/EHMT2 and GLP/EHMT1 are crucial for H3K9 methylation, and their dysregulation has been associated with tumor initiation and progression in different types of cancer. More recently, it has been shown that G9a and GLP appear to play a critical role in several lymphoid hematologic malignancies. Importantly, the key roles played by both enzymes in various diseases made them attractive targets for drug development. In fact, in recent years, several groups have tried to develop small molecule inhibitors targeting their epigenetic activities as potential anticancer therapeutic tools. In this review, we discuss the physiological role of GLP and G9a, their oncogenic functions in hematologic malignancies of the lymphoid lineage, and the therapeutic potential of epigenetic drugs targeting G9a/GLP for cancer treatment.
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SMYD4 is a member of the SMYD family that has lysine methyltransferase function. Little is known about the roles of SMYD4 in cancer. The aim of this study is to investigate genetic alterations in the SMYD4 gene across the most prevalent solid tumors and determine its potential as a biomarker. We performed an integrative multi-platform analysis of the most common mutations, copy number alterations (CNAs), and mRNA expression levels of the SMYD family genes using cohorts available at the Cancer Genome Atlas (TCGA), cBioPortal, and the Catalogue of Somatic Mutations in Cancer (COSMIC). SMYD genes displayed a lower frequency of mutations across the studied tumors, with none of the SMYD4 mutations detected demonstrating sufficient discriminatory power to serve as a biomarker. In terms of CNAs, SMYD4 consistently exhibited heterozygous loss and downregulation across all tumors evaluated. Moreover, SMYD4 showed low expression in tumor samples compared to normal samples, except for stomach adenocarcinoma. SMYD4 demonstrated a frequent negative correlation with other members of the SMYD family and a positive correlation between CNAs and mRNA expression. Additionally, patients with low SMYD4 expression in STAD and LUAD tumors exhibited significantly poorer overall survival. SMYD4 demonstrated its role as a tumor suppressor in the majority of tumors evaluated. The consistent downregulation of SMYD4, coupled with its association with cancer progression, underscores its potential usefulness as a biomarker.
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Mutação , Neoplasias , Humanos , Neoplasias/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Variações do Número de Cópias de DNA , Histona-Lisina N-Metiltransferase/genéticaRESUMO
The current article reports the investigation of three new Ni(II) complexes with ONS-donor dithiocarbazate ligands: [Ni(L1)PPh3] (1), [Ni(L2)PPh3] (2), and [Ni(L2)Py] (3). Single-crystal X-ray analyses revealed mononuclear complexes with a distorted square planar geometry and the metal centers coordinated with a doubly deprotonated dithiocarbazate ligand and coligand pyridine or triphenylphosphine. The non-covalent interactions were investigated by the Hirshfeld surface and the results revealed that the strongest interactions were πâ â â π stacking interactions and non-classical hydrogen bonds C-H···H and C-H···N. Physicochemical and spectroscopic methods indicate the same structures in the solid state and solution. The toxicity effects of the free ligands and Ni(II) complexes were tested on the human breast cancer cell line MCF-7 and non-malignant breast epithelial cell line MCF-10A. The half-maximal inhibitory concentration (IC50) values, indicating that the compounds were potent in inhibiting cell growth, were obtained for both cell lines at three distinct time points. While inhibitory effects were evident in both malignant and non-malignant cells, all three complexes demonstrated lower IC50 values for malignant breast cell lines than their non-malignant counterparts, suggesting a stronger impact on cancerous cell lines. Furthermore, molecular docking studies were performed showing the complex (2) as a promising candidate for further therapeutic exploration.
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Antineoplásicos , Complexos de Coordenação , Simulação de Acoplamento Molecular , Níquel , Humanos , Níquel/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ligantes , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Linhagem Celular Tumoral , Cristalografia por Raios X , Células MCF-7 , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Desenho de FármacosRESUMO
Pancreatic cancer is one of the cancers with very poor prognosis; there is an urgent need to identify novel biomarkers to improve its clinical outcomes. Circulating tumor DNA (ctDNA) from liquid biopsy has arisen as a promising biomarker for cancer detection and surveillance. However, it is known that the ctDNA detection rate in resected pancreatic cancer is low compared with other types of cancer. In this study, we collected paired tumor and plasma samples from 145 pancreatic cancer patients. Plasma samples were collected from 71 patients of treatment-naïve status and from 74 patients after neoadjuvant therapy (NAT). Genomic profiling of tumor DNA and plasma samples was conducted using targeted next-generation sequencing (NGS). Somatic mutations were detected in 85% (123/145) of tumors. ctDNA was detected in 39% (28/71) and 31% (23/74) of treatment-naïve and after-NAT groups, respectively, without referring to the information of tumor profiles. With a tumor-informed approach (TIA), ctDNA detection rate improved to 56% (40/71) and 36% (27/74) in treatment-naïve and after-NAT groups, respectively, with the detection rate significantly improved (p = 0.0165) among the treatment-naïve group compared to the after-NAT group. Cases who had detectable plasma ctDNA concordant to the corresponding tumor showed significantly shorter recurrence-free survival (RFS) (p = 0.0010). We demonstrated that TIA improves ctDNA detection rate in pancreatic cancer, and that ctDNA could be a potential prognostic biomarker for recurrence risk prediction.
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DNA Tumoral Circulante , Neoplasias Pancreáticas , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Neoplasias PancreáticasRESUMO
BACKGROUND: The inhibition of the enzyme telomerase (TERT) has been widely investigated as a new pharmacological approach for cancer treatment, but its real potential and the biochemical consequences are not totally understood. OBJECTIVE: Here, we investigated the effects of the telomerase inhibitor MST-312 on a human glioma cell line after both short- and long-term (290 days) treatments. METHODS: Effects on cell growth, viability, cell cycle, morphology, cell death and genes expression were assessed. RESULTS: We found that short-term treatment promoted cell cycle arrest followed by apoptosis. Importantly, cells with telomerase knock-down revealed that the toxic effects of MST-312 are partially TERT dependent. In contrast, although the long-term treatment decreased cell proliferation at first, it also caused adaptations potentially related to treatment resistance and tumor aggressiveness after long time of exposition. CONCLUSIONS: Despite the short-term effects of telomerase inhibition not being due to telomere erosion, they are at least partially related to the enzyme inhibition, which may represent an important strategy to pave the way for tumor growth control, especially through modulation of the non-canonical functions of telomerase. On the other hand, long-term exposure to the inhibitor had the potential to induce cell adaptations with possible negative clinical implications.
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Antineoplásicos/farmacologia , Benzamidas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Telomerase/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential. METHODS: We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis. RESULTS: Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples. CONCLUSION: The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy.
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DNA Tumoral Circulante/sangue , Neoplasias Colorretais/patologia , Neoplasias Pulmonares/patologia , Plasma/química , Soro/química , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Tumoral Circulante/genética , Neoplasias Colorretais/sangue , Variações do Número de Cópias de DNA , Fragmentação do DNA , Feminino , Frequência do Gene , Rearranjo Gênico , Humanos , Biópsia Líquida , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Background Heterodimeric methyltransferases GLP (EHMT1/KMT1D) and G9a (EHMT2/KMT1C) are two closely related enzymes that promote the monomethylation and dimethylation of histone H3 lysine 9. Dysregulation of their activity has been implicated in several types of human cancer. Patients and methods Here, in order to investigate whether GLP/G9a exerts any impact on Chronic Lymphocytic Leukemia (CLL), GLP/G9a expression levels were assessed in a cohort of 50 patients and the effects of their inhibition were verified for the viability of CLL cells. Also, qRT-PCR was used to investigate the transcriptional levels of GLP/G9a in CLL patients. In addition, patient samples were classified according to ZAP-70 protein expression by flow cytometry and according to karyotype integrity by cytogenetics analysis. Finally, a selective small molecule inhibitor for GLP/G9a was used to ascertain whether these methyltransferases influenced the viability of MEC-1 CLL cell lineage. Results mRNA analysis revealed that CLL samples had higher levels of GLP, but not G9a, when compared to non-leukemic controls. Interestingly, patients with unfavorable cytogenetics showed higher expression levels of GLP compared to patients with favorable karyotypes. More importantly, GLP/G9a inhibition markedly induced cell death in CLL cells. Conclusion Taken together, these results indicate that GLP is associated with a worse prognosis in CLL, and that the inhibition of GLP/G9a influences CLL cell viability. Altogether, the present data demonstrate that these methyltransferases can be potential markers for disease progression, as well as a promising epigenetic target for CLL treatment and the prevention of disease evolution.
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Regulação Leucêmica da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C. METHODS: Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. RESULTS: In patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway. CONCLUSION: Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.
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Metilação de DNA , Epigênese Genética , Epigenômica , Humanos , Contagem de Leucócitos , LeucócitosRESUMO
Approximately 5% of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals will develop one of the HTLV-1-related diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T-cell leukemia. However, the mechanisms responsible for the appearance of symptoms have not been fully clarified. It is believed that viral factors, host genetic and epigenetic mechanisms are implicated in this process. Studies have shown the involvement of histone methyltransferases in retrovirus infection, but no study observed their expression in HTLV-1-infected patients. Among them, euchromatic histone-lysine N-methyltransferase (EHMT)-1 and EHMT-2 were related to retroviral latency in HIV-1 infection. We investigated whether histone methyltransferases EHMT1 and EHMT2 exert any influence on HAM/TSP development by assessing their expression levels in CD4+ T-cells from HTLV-1-infected patients. CD4+ T-cells were immunomagnetically isolated from peripheral blood mononuclear cells of HTLV-1-infected or non-infected individuals and the expression levels of EHMT1 and EHMT2 were determined by RT-qPCR. We observed that EHMT2 was negatively regulated in HTLV-1 asymptomatic carriers compared to non-infected individuals. No difference was observed for EHMT1. These results suggest that EHMT2 downregulation in CD4+ T-cells may be linked to a protection mechanism against the development of HAM/TSP.
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Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical/genética , Paraparesia Espástica Tropical/virologia , Adulto , Linfócitos T CD4-Positivos , Feminino , Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
SET and MYND domain containing 2 (SMYD2) and the SET and MYND domain containing 3 (SMYD3) are the most studied and well-characterized members of SMYD family. It has been demonstrated that their altered expression is associated with the progression of several solid tumors. Nevertheless, whether these methyltransferases exert any impact in chronic lymphocytic leukemia (CLL) remains unknown. Here, we investigated the gene expression profile of SMYD2 and SMYD3 in 59 samples of CLL and 10 normal B cells. The obtained results were associated with white blood cells (WBC) and platelet counts, ZAP-70 protein expression, and cytogenetic analysis. We found that SMYD2 and SMYD3 are overexpressed in CLL patients and, interestingly, patients with residual expression of both genes presented a high WBC count and complex karyotype. Furthermore, a strong correlation between SMYD2 and SMYD3 gene expression was unveiled. Our data demonstrate the association of a residual expression of SMYD2 and SMYD3 with CLL progression indicators and suggests both genes are regulated by a common transcriptional control in this type of cancer. These results may provide the basis for the development of new therapeutic strategies to prevent CLL progression.
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Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Leucemia Linfocítica Crônica de Células B/genética , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
EZH2, a histone methyltransferase, is overexpressed in several human tumors, but whether it exerts any impact in chronic lymphocytic leukemia (CLL) remains unknown. We used real time PCR to investigate the expression profile of EZH1 and EZH2 in 59 CLL patients, 10 samples of purified B-cells from healthy donors and 12 normal adult tissues. EZH2 was overexpressed in CLL patients and correlates with high white blood cell count, ZAP-70 expression and chromosomal abnormalities. EHZ1 expression does not correlate with CLL progression. EZH2 overexpression is related to a poor prognosis of CLL and could be a useful tool to assess its aggressiveness.
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Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/enzimologia , Complexo Repressor Polycomb 2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
Acute Lymphoblastic Leukemia (ALL) is the predominant hematological malignancy in pediatric populations, originating from B- or T-cell precursors within the bone marrow. The disease exhibits a high degree of heterogeneity, both at the molecular level and in terms of clinical presentation. A complex interplay between inherited and acquired genetic alterations contributes to disease pathogenesis, often resulting in the disruption of cellular functions integral to the leukemogenic process. The advent of CRISPR/Cas9 as a gene editing tool has revolutionized biological research, underscoring its potential to modify specific genomic loci implicated in cancer. Enhanced understanding of molecular alterations in ALL has facilitated significant advancements in therapeutic strategies. In this review, we scrutinize the application of CRISPR/Cas9 as a tool for identifying genetic targets to improve therapy, circumvent drug resistance, and facilitate CAR-T cell-based immunotherapy. Additionally, we discuss the challenges and future prospects of CRISPR/Cas9 applications in ALL.
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Telomeres and telomerase are closely linked to uncontrolled cellular proliferation, immortalization and carcinogenesis. Telomerase has been largely studied in the context of cancer, including leukemias. Deregulation of human telomerase gene hTERT is a well-established step in leukemia development. B-acute lymphoblastic leukemia (B-ALL) recovery rates exceed 90% in children; however, the relapse rate is around 20% among treated patients, and 10% of these are still incurable. This review highlights the biological and clinical relevance of telomerase for B-ALL and the implications of its canonical and non-canonical action on signaling pathways in the context of disease and treatment. The physiological role of telomerase in lymphocytes makes the study of its biomarker potential a great challenge. Nevertheless, many works have demonstrated that high telomerase activity or hTERT expression, as well as short telomeres, correlate with poor prognosis in B-ALL. Telomerase and related proteins have been proven to be promising pharmacological targets. Likewise, combined therapy with telomerase inhibitors may turn out to be an alternative strategy for B-ALL.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Telomerase , Criança , Humanos , Telomerase/genética , Telomerase/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Encurtamento do Telômero , Telômero/genética , Telômero/metabolismoRESUMO
In the search for new metal complexes with antitumor potential, two dithiocarbazate ligands derived from 1,1,1-trifluoro-2,4-pentanedione (H2L1) and (H2L2) and four Ni(II) complexes, [Ni(L1)PPh3] (1), [Ni(L1)Py] (2), [Ni(L2)PPh3] (3), and [Ni(L2)Py] (4), were successfully synthesized and investigated by physical-chemistry and spectroscopic methods. The crystal structure of the H2L1 and the Ni(II) complexes has been elucidated by single-crystal X-ray diffraction. The obtained structure from H2L1 confirms the cyclization reaction and formation of the pyrazoline derivative. The results showed square planar geometry to the metal centers, in which dithiocarbazates coordinated by the ONS donor system and a triphenylphosphine or pyridine molecule complete the coordination sphere. Hirshfeld surface analysis by d norm function was investigated and showed π-π stacking interactions upon the molecular packing of H2L1 and non-classical hydrogen bonds for all compounds. Fingerprint plots showed the main interactions attributed to Hâ H Câ H, Oâ H, Brâ H, and Fâ H, with contacts contributing between 1.9% and 38.2%. The mass spectrometry data indicated the presence of molecular ions [M + H]+ and characteristic fragmentations of the compounds, which indicated the same behavior of the compounds in solution and solid state. Molecular docking simulations were studied to evaluate the properties and interactions of the free dithiocarbazates and their Ni(II) complexes with selected proteins and DNA. These results were supported by in vitro cytotoxicity assays against four cancer cell lines, showing that the synthesized metal complexes display promising biological activity.
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Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancer types. Activating oncogenic KRAS mutations are commonly observed in PDAC; however, oncogenic KRAS amplification is rarely observed, and its significance in prognosis and resistance to therapy remains poorly characterized. The present report describes the case of a 52-year-old male patient diagnosed with advanced PDAC with liver metastasis. The patient received modified FOLFIRINOX (mFFX) therapy to which the patient became intolerant with a strong inflammatory response. Subsequent treatment with gemcitabine plus nab-paclitaxel failed to control the disease. Targeted genetic analysis revealed KRAS G12D and TP53 R248Q mutations in the primary tumor and liver metastases. Analysis of circulating tumor DNA (ctDNA) before the first line of treatment confirmed these genetic findings and revealed a >4-fold amplification of the mutant KRAS G12D not detected in the primary tumor. Additionally, subsequent analysis confirmed a 5-fold amplification of the KRAS G12D allele in liver metastasis. Consecutive monitoring of ctDNA revealed an initial decrease in the tumor burden 2 weeks after the first cycle of mFFX. However, coinciding with treatment intolerance, a sharp increase in tumor mutational levels and KRAS G12D amplification was observed 1 month later. The patient died 70 days after treatment initiation. Overall, amplification of oncogenic KRAS G12D was not only associated with an aggressive phenotype, but also supported cancer resistance to chemotherapy. Importantly, this case suggests that plasma detection of KRAS G12D amplification is feasible in the clinical routine and constitutes a powerful tool for assessing tumor aggressiveness.
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Elucidating the conformational heterogeneity of proteins is essential for understanding protein function and developing exogenous ligands. With the rapid development of experimental and computational methods, it is of great interest to integrate these approaches to illuminate the conformational landscapes of target proteins. SETD8 is a protein lysine methyltransferase (PKMT), which functions in vivo via the methylation of histone and nonhistone targets. Utilizing covalent inhibitors and depleting native ligands to trap hidden conformational states, we obtained diverse X-ray structures of SETD8. These structures were used to seed distributed atomistic molecular dynamics simulations that generated a total of six milliseconds of trajectory data. Markov state models, built via an automated machine learning approach and corroborated experimentally, reveal how slow conformational motions and conformational states are relevant to catalysis. These findings provide molecular insight on enzymatic catalysis and allosteric mechanisms of a PKMT via its detailed conformational landscape.
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Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Chromosomal alterations are commonly detected in patients with chronic lymphocytic leukemia (CLL) and impact disease pathogenesis, prognosis, and progression. Telomerase expression (hTERT), its activity and the telomere length are other important predictors of survival and multiple outcomes in CLL. SUV39H and SUV420H enzymes are histone methyltransferases (HMTases) involved in several cellular processes, including regulation of telomere length, heterochromatin organization, and genome stability. Here, we investigated whether SUV39H1, SUV39H2, SUV420H1, SUV420H2, and hTERT are associated with genomic instability of CLL. SUV39H (1/2), SUV420H (1/2), and hTERT expression was determined in 59 CLL samples by real time PCR. In addition, ZAP-70 protein expression was evaluated by Flow Cytometry and patients' karyotype was defined by Cytogenetic Analysis. Low expression of SUV39H1 was associated with the acquisition of altered and complex karyotypes. Conversely, high expression of SUV39H2 correlated with cytogenetic abnormalities in CLL patients. The pattern of karyotypic alterations differed in samples with detectable or undetectable hTERT expression. Furthermore, hTERT expression in CLL showed a correlation with transcript levels of SUV39H2, which, in part, can explain the association between SUV39H2 expression and cytogenetic abnormalities. Moreover, SUV39H1 correlated with SUV420H1 expression while SUV420H2 was associated with all other investigated HMTases. Our data show that the differential expression of SUV39H1 and SUV39H2 is associated with genomic instability and that the modulation of these HMTases can be an attractive approach to prevent CLL evolution. Environ. Mol. Mutagen. 58:654-661, 2017. © 2017 Wiley Periodicals, Inc.
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Instabilidade Genômica/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Linfocítica Crônica de Células B/genética , Metiltransferases/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Cariotipagem , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Telomerase/genética , Telomerase/metabolismo , Telômero/genéticaRESUMO
The aim of the present study was to identify cell types in primary culture from malignant and non-malignant effusions. Effusion samples were subjected to cytology and culture. Immunocytochemistry was performed in cytological slides to evaluate malignancy (positivity for malignancy markers) and in culture slides for identification of cell types in growth. A total of 143 effusion samples (pleural n=76; peritoneal n=37; pericardial n=4; and peritoneal lavage n=26) were analyzed. Cell growth was observed in 34.9% of all samples and immunocytochemistry for identification of cell types in culture slides was conclusive in 90% of them. In non-malignant samples (n=28), growth of mesothelial cells, macrophages and of both cell types was identified in 82.14, 10.71 and 7.14%, respectively. In malignant samples (n=17, all carcinomas), growth of malignant epithelial cells and of both malignant epithelial and mesothelial cells was identified in 41.17 and 23.52%, respectively. In the remaining 35.29% of malignant samples, the only cells in growth were mesothelial and/or macrophages instead of malignant epithelial cells. In conclusion, in culture of malignant effusions, mesothelial cells may be simultaneously identified with malignant epithelial cells. Besides, mesothelial cells and macrophages may be the only cells identified in malignant effusion culture. Therefore, a broad panel of cell markers should be used for unmistakable identification of cells in studies of effusion primary culture. The ideal malignant effusion sample to obtain culture of neoplastic cells should be that without the presence of mesothelial cells and macrophages.