Abstract Cellular senescence is a barrier to tumorigenesis in normal cells, and tumor cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer ...therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning–based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~ 2M lead-like compounds. One hundred and forty-seven virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase assays. Among the found hits, a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced senescence-associated β-galactosidase activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1, and CDC25C. In addition, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long-term treatments. Preliminary structure-activity and structure clustering analyses are reported, and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor–like profile in normal cells, with different pathways affected in cancer cells.
Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; ...however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.
Abstract We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and ...unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.
Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end ...replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.
Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end ...replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.
Cellular senescence has attracted renewed interest in recent years in light of the realization that cancer cells are susceptible to senescence-like responses to stress including exposure to ...chemotherapeutic agents. Therefore, senescence is viewed as a potentially important target in cancer therapy. However, translation of senescence therapeutics will rely on identifying appropriate marker panels for assessing senescence responses in vivo. Although some core pathways governing senescence induction have been clearly identified, the full set of gene targets involved in its establishment and maintenance are not defined and their complex interactions are poorly understood. This view is reinforced by recently discovered roles of microRNA (miRNA) in senescence regulation and induction. Senescence involves coordination of diverse cellular processes ranging from telomere homeostasis and DNA damage to chromatin modifications, inflammatory signaling, and metabolic and cytoskeletal changes. Thus, from a target discovery perspective a "senectome" view is appropriate. miRNAs are well placed to act as distributed senectome control elements, fine-tuning regulation of multiple processes. Here, we examine the complex interactions among several of these subprocesses and subsequently consider senescence-associated miRNAs (SA-miRNAs) as distributed controllers of senectome regulation. Finally, we examine their potential as clinical biomarkers to accelerate development of senescence therapeutics.
Cellular senescence is a barrier to tumorigenesis in normal cells, and tumor cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. ...However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning–based
in silico
screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~ 2M lead-like compounds. One hundred and forty-seven virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase assays. Among the found hits, a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced senescence-associated β-galactosidase activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1, and CDC25C. In addition, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long-term treatments. Preliminary structure-activity and structure clustering analyses are reported, and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor–like profile in normal cells, with different pathways affected in cancer cells.
We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited ...proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in
in vitro
immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.
The generalized inverse Gaussian (GIG) Lévy process is a limit of compound Poisson processes, including the stationary gamma process and the stationary inverse Gaussian process as special cases. ...However, fitting the GIG Lévy process to data is computationally intractable due to the fact that the marginal distribution of the GIG Lévy process is not convolution-closed. The current work reveals that the marginal distribution of the GIG Lévy process admits a simple yet extremely accurate saddlepoint approximation. Particularly, we prove that if the order parameter of the GIG distribution is greater than or equal to −1, the marginal distribution can be approximated accurately — no need to normalize the saddlepoint density. Accordingly, maximum likelihood estimation is simple and quick, random number generation from the marginal distribution is straightforward by using Monte Carlo methods, and goodness-of-fit testing is undemanding to perform. Therefore, major numerical impediments to the application of the GIG Lévy process are removed. We demonstrate the accuracy of the saddlepoint approximation via various experimental setups.
Traditional statistical procedures for estimating the probability of an event result in an estimate of zero when no events are realized. Alternative inferential procedures have been proposed for the ...situation where zero events have been realized but often these are ad hoc, relying on selecting methods dependent on the data that have been realized. Such data‐dependent inference decisions violate fundamental statistical principles, resulting in estimation procedures whose benefits are difficult to assess. In this article, we propose estimating the probability of an event occurring through minimax inference on the probability that future samples of equal size realize no more events than that in the data on which the inference is based. Although motivated by inference on rare events, the method is not restricted to zero event data and closely approximates the maximum likelihood estimate (MLE) for nonzero data. The use of the minimax procedure provides a risk adverse inferential procedure where there are no events realized. A comparison is made with the MLE and regions of the underlying probability are identified where this approach is superior. Moreover, a comparison is made with three standard approaches to supporting inference where no event data are realized, which we argue are unduly pessimistic. We show that for situations of zero events the estimator can be simply approximated with , where n is the number of trials.
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