Senescence as a steppingstone for cancer treatment: If you stop, you might fall

Abstract

This thesis explores the potential of the ‘one-two punch’ therapy, in which cancer cells are first induced to senescence with a first drug, and subsequently targeted by a second (senolytic) drug. In chapter 2, a customized CRISPR-Cas9 genetic screen has been developed to screen for genes that upon knock-out induce senescence. This genetic screen leads to the discovery that ULK1-inhibitors induce senescence in lung, but also in liver, colon, and pancreas cancer cells. Furthermore, it is shown that these senescent cancer cells undergo apoptosis after treatment with ABT-263, a senolytic drug that antagonizes anti-apoptotic proteins. In chapter 3 is sought for common features of senescent cancer cells that would identify universal vulnerabilities and allow unambiguous detection of senescent cancer cells. It appeared that cell lines derived from various cancer types are heterogeneous in their response to ABT-263, their transcriptome, and the SASP factors they express. To find shared expression patterns between the heterogeneous expression patterns of senescent cancer cells, artificial intelligence is used to build the ‘SENCAN classifier model’. This algorithm can predict the probability that cells are senescent using transcriptome data. In chapter 4, the main focus is to find the underlying mechanisms that explain the variability of response to senolytic ABT-263. As ABT-263 acts as an inhibitor of anti-apoptotic proteins, the apoptosis pathway is investigated with BH3 profiling, a technique that measures how close cells are to cell death (apoptotic priming). This technique elucidates that cell lines with a high senolytic response to ABT-263 are more primed to apoptosis, not only in senescent but already in their parental state. Overall, this study shows that senolytic sensitivity is already determined in the parental state, possibly providing a predictive biomarker for the response to senotherapies. In chapter 5, the aim was to identify possible resistance mechanisms to senolytic killing with ABT-263. Where CRISPR/Cas9 resistance screens in proliferating cells rely on the outgrowth of resistant colonies to amplify their gRNA signal, a resistance screen in non-proliferating senescent cells comes with specific adaptations. In this chapter, recommendations are provided on how to perform resistance screens in senescent cells for senolytics. In chapter 6, the research focus is to find a senolytic treatment strategy superior to ABT-263 to kill senescent cancer cells. From a genetic screen, cFLIP is identified as a gene that is essential for the survival of senescent cancer cells. This leads to the finding that senescent cancer cells are more sensitive to the stimulation of extrinsic apoptosis with TRAIL or DR5 agonist in preclinical models. Forthcoming studies will determine whether this therapeutic method will enhance the treatment of cancer patients. In chapter 7, the overall conclusion is that the heterogeneity of senescent cancer cells provides a challenge for the clinical development of the one-two punch strategy, but that various studies also describe various immune-enhancing effects of senescent tumor cells that stimulate tumor killing. Overall, senescence is expected to remain an interesting cancer research area in the coming decades.