Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor ...suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.
Cardiac glycosides are approved for the treatment of heart failure as Na
/K
pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the ...identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.
Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.
At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.
Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.
Abstract
Neuroblastoma, the most common type of pediatric extracranial solid tumor, causes 10% of childhood cancer deaths. Despite intensive multimodal treatment, the outcomes of high-risk ...neuroblastoma remain poor. We urgently need to develop new therapies with safe long-term toxicity profiles for rapid testing in clinical trials. Drug repurposing is a promising approach to meet these needs. Here, we investigated disulfiram, a safe and successful chronic alcoholism treatment with known anticancer and epigenetic effects. Disulfiram efficiently induced cell cycle arrest and decreased the viability of six human neuroblastoma cell lines at half-maximal inhibitory concentrations up to 20 times lower than its peak clinical plasma level in patients treated for chronic alcoholism. Disulfiram shifted neuroblastoma transcriptome, decreasing MYCN levels and activating neuronal differentiation. Consistently, disulfiram significantly reduced the protein level of lysine acetyltransferase 2A (KAT2A), drastically reducing acetylation of its target residues on histone H3. To investigate disulfiram’s anticancer effects in an in vivo model of high-risk neuroblastoma, we developed a disulfiram-loaded emulsion to deliver the highly liposoluble drug. Treatment with the emulsion significantly delayed neuroblastoma progression in mice. These results identify KAT2A as a novel target of disulfiram, which directly impacts neuroblastoma epigenetics and is a promising candidate for repurposing to treat pediatric neuroblastoma.
Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer, accounting for ~25% of all pediatric tumors. Nearly 20% of patients do not respond to current treatments, making ...cALL one of the leading cause of disease-related mortality amongst children. Moreover, sizable portion of childhood leukemia survivors develop significant long-term or late effects of treatment. This rate of refractory/relapse cases and the increased risk of chronic morbidity and early mortality among survivors highlight the pressing need for new treatment strategies in cALL. These can be developed through in-depth investigation of the genetic architecture underlying cALL. For this purpose, we performed whole exome sequencing (WES) of matched tumor-normal pair genomic DNA from 200 cALL patients to identify putative somatic driving mutations. A repertoire of 132 candidate drivers were further validated by performing a targeted knockdown functional screen in 3 pre-B ALL cell lines (NALM6, REH and 697). Through this screen, we identified WHSC1 as putative driver gene. WHSC1 is a methyltransferase that dimethylates lysine 36 on histone H3 (H3K36me2). Eight of the 200 (4%) cALL patients harboured a mutation in WHSC1 (7 pre-B and 1 pre-T ALL). In all 8 cases, the mutation led to a change at amino acid 1099 from a glutamic acid to lysine (E1099K). It has been shown that this activating mutation is associated with global increase in H3K36me2, which is accompanied by substantial changes in gene expression. Transcriptome analysis, using RNA-seq technique, of 6 cALL patients (2 with the mutation and 4 without) revealed distinctive differential expression pattern. We observed a specific enrichment in Wnt pathway genes in presence of the WHSC1 E1099K mutation (6.52 fold, P= 0.05), suggesting a stem cell signature. MCTP39 has been identified as an inhibitor of WHSC1, but has never been tested in ALL. Thus, we treated 4 pre-B ALL cell lines, 3 wild-type for WHSC1 (697, REH and Nalm6) and 1 carrying the mutation E1099K (RS411), as well as the non-leukemic human B cell precursor GM12878 cell line with variable concentrations of MCTP39 and calculated IC50. The IC50 in the leukemic cell lines 697, REH, Nalm6 and RS411 were 2.5µM, 1.7µM, 0.88µM and 0.89µM respectively, whereas it reached 25µM in the non-leukemic GM12878, suggesting a specificity for transformed cells. Although Nalm6 and RS411 had almost the same IC50 at 48h, cell growth curves showed differences in late effect (beyond 3 days), with RS411 being more sensitive to MCTP39. The latter differential effect has been confirmed by colony assay with reduction in plating efficiency of 69% and 45% in RS411 and Nalm6, respectively, when treated 48h with MCTP39 at 0.25µM. This result suggests that MCTP39 targets specifically stem/progenitor cells, particularly those that harbor the mutation WHSC1 E1099K. To confirm that the cellular effect of MCTP39 is due to WHSC1 inhibition, we assessed the impact of MCTP39 on H3K36me2 expression, using fluorometric assay and western blot. In both Nalm6 and RS411cell lines, MCTP39 led to significant decrease in H3K36me2. Then, we analyzed whether MCTP39 synergizes with doxorubicin, an anthracycline that is commonly used in cALL treatment. In both Nalm6 and RS411 cell lines, MCTP39 showed a clear synergistic effect at all concentration tested, permitting 40% reduction in doxorubicin concentration to obtain equal effect. Altogether, this study indicates that inhibition of WHSC1 is a promising treatment strategy in cALL patients, particularly in those carrying WHSC1 mutation. Further investigations are needed to recapitulate these results in vivo and to elucidate the pathways affected as result of the inhibition of H3K36me2 by MCTP39.
No relevant conflicts of interest to declare.
DNA Methylation-Targeted Drugs Da Costa, Elodie M; McInnes, Gabrielle; Beaudry, Annie ...
The cancer journal (Sudbury, Mass.),
2017 Sep/Oct, Volume:
23, Issue:
5
Journal Article
Peer reviewed
Targeting DNA hypermethylation, using nucleoside analogs, is an efficient approach to reprogram cancer cell epigenome leading to reduced proliferation, increased differentiation, recognition by the ...immune system, and ultimately cancer cell death. DNA methyltransferase inhibitors have been approved for the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myelogenous leukemia. To improve clinical efficacy and overcome mechanisms of drug resistance, a second generation of DNA methyltransferase inhibitors has been designed and is currently in clinical trials. Although efficient in monotherapy against hematologic malignancies, the potential of DNA methyltransferase inhibitors to synergize with small molecules targeting chromatin or immunotherapy will provide additional opportunities for their future clinical application against leukemia and solid tumors.
The present study aimed to clarify the role played by the eye/brow and mouth areas in the recognition of the six basic emotions. In Experiment 1, accuracy was examined while participants viewed ...partial and full facial expressions; in Experiment 2, participants viewed full facial expressions while their eye movements were recorded. Recognition rates were consistent with previous research: happiness was highest and fear was lowest. The mouth and eye/brow areas were not equally important for the recognition of all emotions. More precisely, while the mouth was revealed to be important in the recognition of happiness and the eye/brow area of sadness, results are not as consistent for the other emotions. In Experiment 2, consistent with previous studies, the eyes/brows were fixated for longer periods than the mouth for all emotions. Again, variations occurred as a function of the emotions, the mouth having an important role in happiness and the eyes/brows in sadness. The general pattern of results for the other four emotions was inconsistent between the experiments as well as across different measures. The complexity of the results suggests that the recognition process of emotional facial expressions cannot be reduced to a simple feature processing or holistic processing for all emotions.
Purpose: To examine the perceived impacts of an adapted-dance group intervention when added to intensive functional rehabilitation post-stroke.
Method: In this exploratory qualitative case study, ...semi-structured interviews were analyzed using a deliberative inductive logic and referring to the International Classification of Functioning, Disability and Health. Participants were patients in rehabilitation post-stroke (≤25 days) (n = 6), relatives (n = 4) and rehabilitation therapists (n = 12). Selection was on a voluntary basis. The intervention added to the patients' rehabilitation program consisted of 55-minute bi-weekly sessions for 10 weeks.
Results: Three categories of perceived positive impacts emerged from the data: (1) mobility, (2) mental functions and personal factors (emotional functions, motivation and self-efficacy) and (3) interpersonal interactions and social life. A fourth category of perceived impacts involving exercise tolerance was both positive, in terms of general physical endurance, and negative, in terms of a feeling of increased fatigue.
Conclusion: Such an adapted-dance intervention holds promise in subacute rehabilitation post-stroke. Its main strength lies in its perceived positive impact on mental functions, personal factors, and interpersonal and social interactions.
Implications for rehabilitation
An adapted-dance group intervention could offer an innovative means of contributing to intensive functional rehabilitation post-stroke by potentially generating positive perceived impacts on emotional functions, motivation and self-efficacy, as well as on interpersonal and social interactions.
Adapted dance could be added to inpatients' rehabilitation with only minor impacts on fatigue.
Of the basic emotional facial expressions, fear is typically less accurately recognised as a result of being confused with surprise. According to the perceptual-attentional limitation hypothesis, the ...difficulty in recognising fear could be attributed to the similar visual configuration with surprise. In effect, they share more muscle movements than they possess distinctive ones. The main goal of the current study was to test the perceptual-attentional limitation hypothesis in the recognition of fear and surprise using eye movement recording and by manipulating the distinctiveness between expressions. Results revealed that when the brow lowerer is the only distinctive feature between expressions, accuracy is lower, participants spend more time looking at stimuli and they make more comparisons between expressions than when stimuli include the lip stretcher. These results not only support the perceptual-attentional limitation hypothesis but extend its definition by suggesting that it is not solely the number of distinctive features that is important but also their qualitative value.
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