DRUG-SEQ

Targeting iron addiction: Mechanism of action of salinomycin and its synthetic derivative ironomycin against persister cancer cells.

This involves lysosomal iron targeting, production of reactive oxygen species (ROS) and cell death by ferroptosis.

A deeper picture of cancer biology Every individual case of cancer is different, and the chromatin structure inside a cell plays a major role in determining how patients respond to treatment. We spoke to Dr Raphael Rodriguez about his research into how patients respond to anti-cancer drugs, which could lay down the foundations for more personalised treatment in future.

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The effectiveness of cancer treatment

Gene expression

can vary according to the physiology and genetic background of each individual patient, a topic of great interest to researchers. Based at the Institut Curie in Paris, Dr Raphael Rodriguez is a Research Director at the CNRS, a role in which he is probing deeper into cancer biology. “One of the major aims in our research is to better understand the molecular features of each patient, starting from their genetic and epigenetic features,” he outlines. The structure of chromatin, a component of eukaryotic cells, which is comprised of DNA and proteins, is an important consideration in that respect. “The DNA sequence is a major factor in the structure of chromatin,” explains Dr Rodriguez. “A second point is the histones, proteins around which DNA is wrapped. These histones can be chemically modified, and it is these modifications that essentially determine how the DNA is packed and whether it is accessible to other proteins.”

This helps to determine which genes are expressed or repressed and the defects that may be present in a cell, important considerations in terms of Dr Rodriguez’s wider research agenda. Some existing anticancer drugs target proteins that interact with DNA, or proteins that interact with chromatin, and the effectiveness of these approaches will depend to a degree on how chromatin is structured. “The response of individual patients will be different. This is what we are trying to understand,” says Dr Rodriguez. Cancer is often very heterogeneous in terms of cell types however, which adds another layer of complexity to Dr Rodriguez’s research. “We know that using one drug for a patient doesn’t work, because there are different types of cells and different types of defects,” he continues. “What we can do is try to identify the different types of cells and treat them with a combination of drugs.”

A major challenge here is to understand the heterogeneity of a particular cancer, which could then inform clinical decisionmaking and lead to improved outcomes for patients. Researchers in Dr Rodriguez’s group are using DNA sequencing to identify an individual’s genetic and epigenetic features, which will help clinicians choose the most effective drug for that particular patient.

“That’s one aspect of what we are trying to achieve,” he explains. This research also holds relevance to understanding cancer resistance; for many years researchers struggled to identify the different types of cells in cancer, failing to recognise that some of the cells in a tumour do not divide as quickly as others. “That’s quite a natural thing to do in a way. When you use an antiproliferative drug and see that the tumour is shrinking then that’s a positive outcome,” points out Dr Rodriguez. “What you do not know is whether the remaining cells are dividing aggressively, and what’s going to happen when you stop this treatment.”

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Many cancers are or acquired the potential to become resistant, and ‘persister’ cancer cells are thought to be major actors underlying cancer relapse. Cancer stem cells are so named because they have some features and characteristics related to those that can be seen in stem cells, in particular the ability to self-renew and to seed new tumour tissues. “A single cancer stem cell or small group of these cells can generate an entire tumour from these cell, which is not the case with a normal cancer cell,” says Dr Rodriguez. It is thought that around 1-2 percent of the cells inside solid tumours exhibit these stem cell properties, which Dr Rodriguez says gives them the capacity to leave the site of a primary tumour and play a role in the development of a secondary tumour. “They can migrate and contribute to metastasis. This is something that normal cancer cells are not capable of doing,” he explains. “These cancer stem cells also do not typically divide as quickly as we would like them to.”

A cancer cell’s identity is not fixed however and it can become a cancer stem cell, another topic of interest to Dr Rodriguez and his colleagues. Researchers are investigating how cells can shift between an epithelial, proliferative state, to a mesenchymal, less proliferative, but more migratory and invasive state. “Most

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of the migratory or invasive capacity of epithelial cells is reduced,” outlines Dr Rodriguez. During the course of their research, Dr Rodriguez and his colleagues have discovered that the transition between the epithelial and mesenchymal states is regulated by the presence of iron.

“Iron is required to change the behaviour of cancer cells. We are looking at how metals can basically unlock the expression of certain genes involved in migration,” he continues. “We’re looking at the role of iron inside the nucleus of cells, at how it essentially unlocks the expression of genes involved in the mesenchymal state of cells.”

Iron uptake The next step beyond this could be to block the uptake of iron inside these cells, a possibility that Dr Rodriguez is exploring.

When iron is in these cells it is transported via an organelle called the lysosome before eventually reaching the nucleus, a process that researchers are looking to disrupt.

“We are developing new molecules that can block iron uptake and trafficking in cells. These molecules can interfere with translocation from the lysosome, such that the iron would no longer go to the nucleus and contributes instead to toxic oxidative stress, killing these cells through a mechanisms reminiscent to ferroptosis,” Cellular detection of lysosomal iron(II) in cancer cells.

Iron Lysosomes Cell Nucleus

Iron

Lysosomes

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Drug-Seq Unravelling the Genomic Targets of Drugs Using High-Throughput Sequencing

Project Objectives

1. Defining where small molecules bind in genomes.

2. Defining the epigenetic alterations as a results of targeting lysosomal iron.

Lysosomal iron targeting in persister cancer cells, regulation of iron endocytosis via CD44 and hyaluronic acids, iron-regulation of epigenome in cancer.

Project Funding

(Total funding over € 4.5M). ERC consolidator, Labellisation Ligue Contre le Cancer, Fondation Charles Defforey, Emergence Ville de Paris, FRM, National Health & Medical Research Council of Australia.

Project Partners

• Kyle Miller • Emmanuelle Charafe-Jauffret • Christophe Ginestier • Mark Dawson

Contact Details

Raphaël Rodriguez, Ph.D., FRSC Research Director at the CNRS Head of Chemical Biology of Cancer CNRS UMR3666 - INSERM U1143 Institut Curie 26 rue d’Ulm Pav. Trouillet 75005 Paris France T: +33 648 482 191 E: raphael.rodriguez@curie.fr W: https://science.institut-curie.org/team-rodriguez CD44 regulates epigenetic plasticity by mediating iron endocytosis. Sebastian Müller, Fabien Sindikubwabo, Tatiana Cañeque, Anne Lafon, Antoine Versini, Christophe Ginestier, Emmanuelle Charafe-Jauffret, Bérangère Lombard, Damarys Loew, Ting-Di Wu, Adeline Durand, Céline Vallot, Sylvain Baulande, Nicolas Servant, Raphaël Rodriguez*. bioRi doi: https://doi.org/10.1101/693424.

Salinomycin kills cancer stem cells by sequestering iron in lysosomes. T. T. Mai, A. Hamaï, A. Hienzsch, T. Cañeque, S. Müller, J. Wicinski, O. Cabaud, C. Leroy, A. David, V. Acevedo, A. Ryo, C. Ginestier, D. Birnbaum, E. Charafe-Jauffret, P. Codogno, M. Mehrpour*, R. Rodriguez* Nature Chem. 9, 1025-1033 (2017). Highlighted in CEN.

says Dr Rodriguez. This work builds on the discovery their in-house small molecule called ironomycin alters gene expression profile by targeting lysosomal iron. “When we discovered that ironomycin and the parental natural product salinomycin were selectively killing cancer stem cells by targeting iron, we hypothesised that these cells contained more iron,” explains Dr Rodriguez. “It seemed that these cells, in order to stay in a stem state, needed more iron. We did indeed find that they had an increased iron load.”

epigenetic marks that are affected by iron, valuable information in the context of the search for more personalised forms of cancer treatment.

This would be complemented by drugs that are more specific to each patient, recognising that each individual case is different. The development of new drugs and therapies forms a major part of Dr Rodriguez’s research agenda. “We work at the crossroads of chemistry and biology and we are seeking to develop effective drugs. We are in close contact with basic

“We work at the crossroads of chemistry and biology and we are seeking to develop effective drugs.

We are in close contact with medical doctors here at Institut Curie.

We have developed new drug-like molecules, to target the iron metabolism in cancer.”

A major goal here is to understand how these cells use iron in order to maintain their stem cell properties. Researchers used a combination of techniques, including quantitative proteomics, quantitative metabolomics, next generation DNA sequencing, and next generation RNA sequencing to look at these cells in great depth, and Dr Rodriguez says this work has yielded some new insights. “We were able to highlight a few proteins that use iron to produce particular metabolites that lead to the depletion of some histone markers that regulate the shape of chromatin,” he outlines. The aim is to identify the

research scientists and medical doctors here at Institut Curie. We have developed new drug-like molecules, to target iron metabolism in cancer,” he says. A biotech company has been established to look into bringing these new drugs to the clinic, yet Dr Rodriguez is keen to stress that he will continue with more exploratory research into fundamental mechanisms in cancer chemistry and biology in future. “Academic research is very important because we generate knowledge that has intrinsic value in its own sense. Alongside that we are also developing new compounds,” he continues.

Raphaël Rodriguez

Raphaël Rodriguez is the Marie Curie Chair of Chemical Biology at the Institute Curie, where he holds a Senior Principal Investigator Position. He gained his PhD training from the University of Marseille (France) and Oxford (UK), where he completed the total synthesis of complex natural products. He is the first French citizen to receive the prestigious Tetrahedron Young Investigator Award and is the Cofounder of the biotech company SideROS.

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