In the past 5 years, the laboratory of Agami used ribosome profiling technology to map with nucleotide precision ribosome position on mRNAs. This technology allows studying events regulated at the mRNA translation level. They initially exploited this technology in the study of the tumour suppressor p53, translation in the mitochondria, and in response to oncogenic Myc activation. However, the most significant application of this technology was its use for the discovery of amino acid shortages in cancer cells (Loayza-Puch. et al., Nature 2016).
The demand for amino acids for protein, nucleotide and energy production, as well as for balancing oxidative stress, is very high in the growing tumour. To survive and flourish, tumours require diverting amino acid pathways to adapt to their deregulated and recurrent growth, forming
metabolic changes that can be exploited as vulnerabilities for therapy. An example for such a vulnerability is the amino acid asparagine in acute lymphoblastic leukaemia. Asparagine depletion by the administration of L asparaginase results in a very effective combined treatment with chemotherapy (~95% cure).
The agami lab developed an innovative measurement approach to determine restrictive amino acids in cells and named it diricore (differential ribosome codon reading). Using diricore they uncovered a shortage in proline in breast cancer and kidney tumours. Currently the lab exploits proline shortage for cancer therapy by means of restricting its availability to tumours. In 2017, in a follow up study they applied diricore to a cellular model of metastasis and aggressive tumour behaviour, and uncovered shortage of leucine (Loayza-Puch et al., EMBO Reports 2017). Ongoing experiments investigate amino acid shortages in various cancer contexts. Granted subsidies from the Dutch cancer Society allows further investigation along these lines of research.
Enhancers are non-coding genomic regions that activate gene expression of distantly located target genes. Interestingly, genes encoding enhancer- binding proteins were linked to cancer development, and novel cancer therapies block enhancer function. However, the human genome is estimated to contain up to a million different enhancers while only few are potentially involved. The Agami lab studied the tumour suppressor p53, and showed that it binds enhancers that produce enhancer RNAs (eRNAs). They showed, for the first time, that p53-induced eRNAs are required for enhancer activity and p53 function (Melo et al., Molecular Cell 2013).
Intriguingly, recent studies have indicated that single nucleotide polymorphisms, large-scale genomic rearrangements, and somatic mutations, can affect enhancer activity and by that contribute to tumour development and it' s aggressiveness. However, systematic identification of enhancer functions was hampered by the lack of tools to perform unbiased functional genetic screens.
The Agami lab therefore devised a novel approach for this purpose by utilizing the genome editing CRISPR tool. They presented two proof-of–concept genetic screens to identify and characterize functional enhancers in their native environment. This resulted in the identification of potential tumour suppressive and oncogenic enhancers that are targets of p53 and estrogen receptor.
Additionally, they showed that this technology is suitable for de novo identification of novel enhancer elements using a genomic tiling approach (Korkmaz et al., Nature Biotech. 2016; Elkon et al., Nature Biotech. 2017; Han et al., Genome Biology 2018, Lopes et al., Cancer letters 2018 ). Altogether, the Agami lab paved novel ways to expand the utility of the CRISPR tool to explore in an unbiased manner the functions of the non-coding genome under normal and pathological conditions. A granted subsidy from the Dutch government allows the Agami group to explore this technology further and gain a more comprehensive view of enhancer functions in cancer.
Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. The Agami team suspected a connection between transcription and translation.
Interestingly, genome- wide analyses and in vitro experiments supported such a link between the
rate of transcription and the efficacy of translation. Moreover, the group identified m6A modification of mRNAs as a mediator of this connection.
This study uncovered a general and widespread link between transcription and translation that is governed by epigenetic mRNA modifications (Slobodin et al., Cell 2017).
The ends of most message RNAs (mRNAs) are cleaved and
polyadenylated, a process that is required for mRNA function. Recent discoveries revealed that a large proportion of human genes contain more than one polyadenylation site. Therefore, alternative cleavage and polyadenylation (APA) is a widespread phenomenon that generates mRNAs with alternative 3’ ends.
Potentially, APA generates isoforms differing in their coding and non-coding regions and provides an important regulatory layer of gene expression. However, whether and how APA is regulated was largely unknown.
The Agami lab developed a reporter- based RNAi screen and devised a genome-wide approach to detect 3’- ends of mRNAs. With these tools, they identified the gene PABPN1 as a regulator of APA (Jenal et al., Cell 2012). Interestingly mutations in PABPN1 causes the autosomal dominant oculopharyngeal muscular dystrophy (OPMD).
As predicted from their experiments, indeed the expression of mutant PABPN1 in both a mouse model of OPMD and human cells elicited 3’-UTR shortening, linking for the first time APA with a genetic disease. Additionally, the team explored, on a transcriptome-wide scale, APA events that are associated with cancer and heart disease (Elkon et al., Genome Biol. 2012; Morris et al., Clin. Cancer Res. 2012; Elkon et al., Nature Rev. Genetics 2013; Creemers et al., Circ. Res. 2016; Hoffman et al., PLOS Genet. 2016).