The Expression and Activation of the NF-κB Pathway Correlate with Methotrexate Resistance and Cell Proliferation in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although its prognosis continually improves with time, a significant proportion of patients still relapse from the disease because of the leukemia's resistance to therapy. Methotrexate (MTX), a folic-acid antagonist, is a chemotherapy agent commonly used against ALL and as an immune-system suppressant for rheumatoid arthritis that presents multiple and complex mechanisms of action and resistance. Previous studies have shown that MTX modulates the nuclear factor kappa B (NF-κB) pathway, an important family of transcription factors involved in inflammation, immunity, cell survival, and proliferation which are frequently hyperactivated in ALL. Using a gene set enrichment analysis of publicly available gene expression data from 161 newly diagnosed pediatric ALL patients, we found the Tumor necrosis factor α (TNF-α) signaling pathway via NF-κB to be the most enriched Cancer Hallmark in MTX-poor-responder patients. A transcriptomic analysis using a panel of ALL cell lines (six B-cell precursor acute lymphoblastic leukemia and seven T-cell acute lymphoblastic leukemia) also identified the same pathway as differentially enriched among MTX-resistant cell lines, as well as in slowly dividing cells. To better understand the crosstalk between NF-κB activity and MTX resistance, we genetically modified the cell lines to express luciferase under an NF-κB-binding-site promoter. We observed that the fold change in NF-κB activity triggered by TNF-α (but not MTX) treatment correlated with MTX resistance and proliferation across the lines. At the individual gene level, NFKB1 expression was directly associated with a poorer clinical response to MTX and with both an increased TNF-α-triggered NF-κB activation and MTX resistance in the cell lines. Despite these results, the pharmacological inhibition (using BAY 11-7082 and parthenolide) or stimulation (using exogenous TNF-α supplementation) of the NF-κB pathway did not alter the MTX resistance of the cell lines significantly, evidencing a complex interplay between MTX and NF-κB in ALL.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Introduction: Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. Methods: To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. Results: MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Conclusion: Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

Germline variants in DNA repair genes associated with hereditary breast and ovarian cancer syndrome: analysis of a 21 gene panel in the Brazilian population.

BACKGROUND: The Hereditary Breast and Ovarian Cancer Syndrome (HBOC) occurs in families with a history of breast/ovarian cancer, presenting an autosomal dominant inheritance pattern. BRCA1 and BRCA2 are high penetrance genes associated with an increased risk of up to 20-fold for breast and ovarian cancer. However, only 20-30% of HBOC cases present pathogenic variants in those genes, and other DNA repair genes have emerged as increasing the risk for HBOC. In Brazil, variants in ATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN, and TP53 genes have been reported in up to 7.35% of the studied cases. Here we screened and characterized variants in 21 DNA repair genes in HBOC patients. METHODS: We systematically analyzed 708 amplicons encompassing the coding and flanking regions of 21 genes related to DNA repair pathways (ABRAXAS1, ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53 and UIMC1). A total of 95 individuals with HBOC syndrome clinical suspicion in Southeast Brazil were sequenced, and 25 samples were evaluated for insertions/deletions in BRCA1/BRCA2 genes. Identified variants were assessed in terms of population allele frequency and their functional effects were predicted through in silico algorithms. RESULTS: We identified 80 variants in 19 genes. About 23.4% of the patients presented pathogenic variants in BRCA1, BRCA2 and TP53, a frequency higher than that identified among previous studies in Brazil. We identified a novel variant in ATR, which was predicted as pathogenic by in silico tools. The association analysis revealed 13 missense variants in ABRAXAS1, BARD1, BRCA2, CHEK2, CDH1, MLH1, PALB2, and PMS2 genes, as significantly associated with increased risk to HBOC, and the patients carrying those variants did not present large insertions or deletions in BRCA1/BRCA2 genes. CONCLUSIONS: This study embodies the third report of a multi-gene analysis in the Brazilian population, and addresses the first report of many germline variants associated with HBOC in Brazil. Although further functional analyses are necessary to better characterize the contribution of those variants to the phenotype, these findings would improve the risk estimation and clinical follow-up of patients with HBOC clinical suspicion.

Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia.

Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of ß-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells.

Synthesis and evaluation of 2-carboxy indole derivatives as potent and selective anti-leukemic agents.

Despite the success achieved in the treatment of acute lymphoblastic leukemia (ALL), the search for new drugs featuring selectivity against leukemia cells and effectiveness to prevent relapsed ALL is still highly desirable. Here, we described the synthesis of several novel 3-substituted and 3,6-disubstituted-2-carboalkoxy indoles followed by the elucidation of their mechanism of action and in vivo anti-leukemia efficacy. The synthesis of 3-substituted-2-carboalkoxy indoles relied on two Heck arylations of methyl acrylate and methyl cinnamates respectively, to generate ß,ß-disubstituted acrylates followed by an efficient Cadogan-Sundberg reaction of these latter intermediates. The method developed led to the synthesis of twenty-one novel functionalized indoles. Of these, indole 20 showed selective cytotoxicity against leukemia cells at the nanomolar scale, and, therefore, it was selected for the investigation of its mechanism of action. Indole 20 was found to target tubulin leading to G2/M cell cycle arrest, DNA damage and apoptosis. Indole 20 decreased ß-tubulin protein in leukemia cells in a time-dependent manner and induced depolymerization of the microtubule network in Hela cells, thus fully characterizing its microtubule destabilizer activity. The connectivity map analysis of HL60 promyelocytic leukemia cells treated with indole 20 revealed a transcriptional profile similar to that of cells treated with prostaglandins, apparently due to the induction of cellular differentiation as addressed by the expression of CD11 and CD14 markers. Finally, indole 20 given intraperitoneally, at 10 mg/kg, 5x/week significantly prolonged the overall survival of NOD/SCID mice transplanted with RS4; 11 B-ALL cells.

Up-regulation of SPINT2/HAI-2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion.

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS- and de novo AML-BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI-2 levels up-regulation. Moreover, Aza treatment of HD-BMMSC did not improve SPINT2/HAI-2 levels. To understand the role of SPINT2/HAI-2 down-regulation in BMMSC physiology, SPINT2/HAI-2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS-5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI-2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI-2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down-regulation in SPINT2/HAI-2 gene expression, due to methylation in MDS-BMMSC and de novo AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.

Low Bioavailability and High Immunogenicity of a New Brand of E. colil-Asparaginase with Active Host Contaminating Proteins.

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance.

The Ala55Val and -866G>A polymorphisms of the UCP2 gene could be biomarkers for weight loss in patients who had Roux-en-Y gastric bypass.

OBJECTIVE: The aim of this study was to investigate whether the Ala55Val and -866G>A polymorphisms of the UCP2 gene are related to weight loss and changes in body composition after bariatric surgery performed by Roux-en-Y gastric bypass (RYGB). METHODS: This longitudinal study enrolled obese patients submitted to RYGB. Data regarding weight (kg), body mass index (kg/m2), fat-free mass (FFM; kg), fat mass (kg), weight loss (kg and %), and percent excess weight loss were collected from both preoperative and 1-y postoperative medical records. Polymorphisms were genotyped by allelic discrimination using real-time polymerase chain reaction and TaqMan-predesigned single nucleotide polymorphism Genotyping Assay kits (Applied Biosystems, Foster City, CA, USA). The t test was used to compare variables between genotypes of each polymorphism to analyze the dominant and recessive models. Linear regression models were used to adjust the effects of initial weight, age, and sex on the variation of weight and body composition (P < 0.05). RESULTS: We analyzed 150 severely obese individuals (age 47.2 ± 10.5 y; 80% women). Genotype analysis showed a greater prevalence of heterozygous GA (41.3%) for -866G>A polymorphism and CT (39.3%) for Ala55Val polymorphism. Individuals who carried the T (CT+TT) and A (GA+AA) mutated alleles for Ala55Val and -866G>A, respectively, showed a higher weight and FFM loss. CONCLUSION: The mutated alleles T for Ala55Val and A for -866G>A polymorphism could be biomarkers of weight loss 1 y after RYGB.

Polymorphisms of the CYP1A1 and CYP2E1 genes in head and neck squamous cell carcinoma risk.

Polymorphisms in genes that encode P450 cytochrome enzymes may increase carcinogen activation or decrease their inactivation and consequently, promote the development of cancer. The aims of this study were to identify the MspI-CYP1A1, PstI-CYP2E1 and DraI-CYP2E1 polymorphisms in patients with head and neck cancer and to compare with individuals without cancer; to evaluate the association of these polymorphisms with risk factors and clinical histopathological parameters. In the study group, 313 patients were evaluated for CYP1A1, 217 for CYP2E1 (PstI) and 211 for CYP2E1 (DraI) and in the control group 417, 334 and 374 individuals, respectively. Molecular analysis was performed by PCR-RFLP technique, and chi-square and multiple logistic regression tests were used for statistical analysis. The result of analysis regarding individuals evaluated for CYP1A1 (MspI) showed that age (OR: 8.15; 95% CI 5.57-11.92) and smoking (OR: 5.37; 95% CI 3.52-8.21) were predictors for the disease; for the CYP2E1 (PstI and DraI), there were associations with age (PstI-OR: 9.10; 95% CI 5.86-14.14/DraI-OR: 8.07; 95% CI 5.12-12.72), smoking (PstI-OR: 4.10; 95% CI 2.44-6.89/DraI-OR: 5.73; 95% CI 3.34-9.82), alcohol (PstI-OR: 1.93; 95% CI 1.18-3.16/DraI-OR: 1.69; 95% CI 1.02-2.81), respectively, with disease development. CYP2E1 (PstI) was less frequent in patient group (OR: 0.48; 95% CI 0.23-0.98). Regarding clinical histopathological parameters, CYP1A1 polymorphism was less frequent in the larynx primary anatomic site (OR = 0.45; 95% CI = 0.28-0.73; P = 0.014). In conclusion, we confirm that age, smoking and alcohol consumption are risk factors for this disease and the polymorphisms investigated have no association with the development of head and neck cancer.