Identification of novel modulators of protein synthesis and nucleolar biology using high throughput phenotypic screens

Abstract

Protein synthesis and ribosome biogenesis are fundamental steps in gene expression and constitute the most energy demanding processes in living cells. Dysregulation of these processes is associated to a variety of human disorders including cancer, metabolic diseases, immunodeficiency, neurological and developmental disorders, and physiological aging. Therapeutic strategies modulating protein synthesis and ribosome biogenesis or nucleolar biology, have proven to be efficient for several of these disorders, and some of them are already used in the clinic, predominantly in the context of cancer. However, the success of these drugs has been limited due to activation of mechanisms of resistance or lack of general effects among different cancer types. Additionally, the application of modulators of protein and ribosome production in other disease contexts is just starting to be explored. This is particularly important for disorders where altered translation control is a hallmark, such as in the case of some neurodegenerative diseases. Moreover, different disorders may require different therapeutic approaches, hence, research in less known disease areas opens possibilities of finding new ways of regulating protein synthesis and ribosome biogenesis, and perhaps new biology.

In this thesis we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology. Phenotypic screening allows for the systematic identification of regulators of an organismal feature (phenotype) without having any prior knowledge.

In paper I we benefited from novel technologies allowing visualization of changes in protein synthesis to evaluate the effects of medically approved and well-characterized drugs in mRNA translation. Our screen failed to identify small molecules stimulating translation in cancer cells growing in complete media. Yet, it seems that translation can only be boosted when the translation machinery of cells is challenged, such as when cells are grown under starvation conditions. Nevertheless, our screen identified known down-regulators of translation, supporting the validity of our approach, and a new translation inhibitor, SKI-II. SKI-II was developed as a sphingosine kinase inhibitor (SPHK), and this group of compounds has been explored extensively as anticancer drugs. However, in our hands, SKI-II inhibited translation by inducing the integrated stress response (ISR), causing physical damage to the endoplasmic reticulum (ER), which resulted in cell death. The toxicity of SKI-II and its clinically relevant analog ABC294640 was not abrogated when knocking out sphingosine kinases, while it was partially rescued upon inhibition of the ISR. Our work is the first to systematically examine the effect of known drugs in translation in cells and to report cytotoxic properties of SPHK inhibitors that are independent of SPHKs.

In paper II we conducted a chemical screen to identify compounds limiting the toxicity of amyotrophic lateral sclerosis (ALS)-related dipeptide repeats (DPRs). ALS is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, leading to muscular paralysis and death, within 3 to 5 years after diagnosis. The expansion of G4C2 repeats within the first intron of the C9ORF72 gene constitutes the most common cause of ALS and frontotemporal dementia (FTD). Through repeat-associated non-ATG (RAN) translation, these expansions are translated into DPRs, some of which, poly-proline arginine (PR) and poly-glycine arginine (GR), bind to the nucleoli and lead to cell death. Here we conducted a screen to identify compounds reducing toxicity of twenty-repeats poly-PR peptides (PR20) added exogenously to cells in culture. Our screen identified two BET bromodomain inhibitors (Bromosporine-1 and PFI-1) and sodium phenylbutyrate (Na-Phen), currently in clinical trials, as modifiers of PR20 toxicity in different cell lines and in developing zebrafish embryos. Our work shows that BET Bromodomain inhibitors rescue the nucleolar stress induced by PR20 and the known nucleolar stressor Actinomycin D (ActD). To our knowledge, this is the first time that compounds able to protect nucleolar integrity are reported in the literature, and therefore, they might have beneficial effects in diseases associated to nucleolar stress, such as ALS/FTD.

Inspired by our results, we conducted four additional screens that are collected in the section preliminary results. Following paper I, we applied the same screening pipeline to identify novel modulators of translation among natural compounds (preliminary results I). Related to paper II, the literature points to two main issues with current modulators of ribosome biogenesis, promiscuity, even among the so-called selective modulators, and heterogeneity in the efficacy of compounds across different cancer types. Regarding the first, the discovery of regulators of ribosome biogenesis has advanced in parallel with the technology allowing their study. Current methods allow better characterization of the activities of these drugs and development of strategies to find more selective modulators, which we reviewed in annex I. Nevertheless, there is a growing need for novel modulators of nucleolar activity, and we benefited from publicly available image datasets to explore the effects of known drugs in the nucleolus (preliminary results II). Also, we conducted a genome-wide CRISPR/Cas9 screen to identify vulnerabilities to nucleolar stressors and systematically interrogate in which genetic backgrounds these drugs are suitable anticancer therapies (preliminary results III). Lastly, triggered by the discovery of “nucleolar protectors” in paper II, we conducted a chemical screen to explore novel nucleolar functions of known drugs using the Drug Repurposing Hub library 1 from the Broad Institute (preliminary results IV).

Altogether, here we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology relevant for disease contexts, and to uncover new biology linked to these processes.