Unravelling the Genomic Targets of Drugs Using High-Throughput Sequencing

Projektinformationen

Drug-Seq

ID Finanzhilfevereinbarung: 647973

DOI

10.3030/647973

Projekt abgeschlossen

EK-Unterschriftsdatum 20 Juli 2015

Startdatum 1 September 2015

Enddatum 28 Februar 2022

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 1 999 900,00

EU-Beitrag

€ 1 999 900,00

Koordiniert durch

INSTITUT CURIE
France

Periodic Reporting for period 4 - Drug-Seq (Unravelling the Genomic Targets of Drugs Using High-Throughput Sequencing)

Berichtszeitraum: 2020-03-01 bis 2022-02-28

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

1. What is the problem/issue being addressed?
The clinical management of cancers involves the use of drugs including small molecules that target genomic DNA, to elicit DNA damage and cell death.
The efficacy of these treatments varies between cancers, between patients, and evolve during treatment.
It is, as yet, no entirely clear why or how cancer cells evolve to become refractory to genotoxic drugs.
An interesting work hypothesis is that chromatin, the complex between genomic DNA and histone proteins, defines genomic sites of action of these drugs.
Given that the chromatin status varies between cell lines and individuals, it would explain, at least in part, how cancer cells modify chromatin structure to become refractory to these drugs.

2. What is the problem/issue being addressed?
Technologies designed to assess genome targeting with small molecules including DNA sequencing and cell imaging, could be highly valuable.
In particular, these technologies may help delineate mechanisms of action in a patient-dependent manner (e.g. help assess variability of drug effects between samples), and hopefully help predict drug responses.

3. What are the overall objectives?
The objectives of the program are to design and synthesize clickable small molecules and develop molecular-based strategies to visualize genotoxic and other drugs in cells post-treatment by fluorescence microscopy and/or to pull-down genomic targets to be subjected to deep sequencing.
We anticipate that data gathered from these technologies will help understand, predict and rewire drug responses, providing the means for personalized medicine.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

As part of our proposed research, we had initially planned to mainly work on three genotoxic compounds including etoposide, camptothecin and cisplatin.

1. Etoposide
Prior work from our group on etoposide led to the development of topoisomerase II beta isoform analogues of etoposide. This work could potentially provide the basis for future ERC-funded investigations.

2. Camtothecin
We have also developed a clickable derivative of topotecan that is biologically active and in principle suitable for cell imaging and DNA sequencing using click chemistry.

3. Cisplatin
We have successfully developed two platinum-derived drugs named APPA and APPOA. These drugs are are derived from the clinically approved picoplatin and oxaliplatin.
These two analogues are biologically active and suitable for click chemistry. We have successfully used these two analogues and develop new protocols to visualize platinated DNA lesions (DNA-Pt) in various commercially available cancer cell lines.

During the course of this program, we have also investigated three other classes of drugs to broaden the scope of the program.

4. JQ1
The BET-Bromodomain inhibitor JQ1. In collaboration with Mark Dawson (Peter Maccallum Cancer Center, Melbourne) and GSK, we have developed a biologically active clickable analogue of JQ1 that is suitable for cell imaging, FACS analysis, protein pull-down and DNA sequencing (Click-Seq).


5. Salinomycin
This natural product has been shown to target cancer stem cells, the population of cells that is refractory to conventional treatments, that promotes metastasis and relapse. The anti-cancer stem cell mechanism of Salinomycin is unknown. Furthermore, this natural product has been shown to induce DNA damage. We have developed a clickable analogue and developed protocols to visualize this new probe in cells. We have also synthesized a series of other clickable derivatives that are more potent and more selective towards cancer stem cells.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

We have made a series of discoveries that go beyond state of the art using the small molecule probes we have developed.

1. Cisplatin
We used APPA as surrogates of cisplatin to visualize DNA-Pt in cells with high resolution. We discovered that the histone deacetylase inhibitor SAHA treatment leads to the production of clusters of DNA-Pt that recruit and become resistant to the lesion bypass and resistance machinery translesion synthesis. We have shown that it was possible to reprogram a resistance mechanism into an apoptotic trigger. This is the first demonstration that targeting chromatin sensitize the genome to a genotoxic agent (1 Paper + 1 Patent).

2. Marmycin was discovered as a DNA damaging agent. Because of the scarce nature of the natural source, we established a synthetic route. We discovered that it was eliciting DNA damage through lysosomal targeting. This has led the general conceptualization that compounds can accumulate in lysosome and trigger alterations at the chromatin level indirectly, thereby shifting the scope of DRUG-seq (1 Paper in Nat Chem).

3. JQ1
We have used our clickable JQ1 analogue (JQ1-PA) to visualize this drug in cells and in tissues for the first time. In vivo aspects of the project was performed by M. Dawson and GSK. Interestingly, we have shown that it was possible to correlate drug staining in cells with alteration of gene expression profiles induced by this drug. This work provide the basis for future personalized medicine based on epigenetic treatments (1 publication in Science).

4. Salinomycin
Using click chemistry, we have discovered that our natural product surrogate we named Ironomycin, targets and sequesters iron(II) in the lysosomal compartment of cancer stem cells. We have discovered that cancer stem cells up-regulate iron homeostasis (1 publication in Nat Chem). These unprecedented findings provide additional basis for personalized medicine using iron as a marker of relapse or using iron depletion to sensitize cancer cells to other genotoxic agents. This work is currently being pursued in our laboratory.

5. The finding that salinomycin exerts it activity through the targeting of lysosomal iron has provided solid evidence that persister cancer cells are addicted to iron and identified lysosomal iron as primary source of ferroptosis, which has so far remained elusive. This work now provides the basis for the development of next generation therapeutics that can trigger ferroptosis in the persister cancer cell state (2 publication in Cancer Discovery 2022, 3 concept published in Mol Cell 2022). We have shown that through the retention of iron in lysosome, ironomycin induces a nuclear iron depletion that directly affects the epigenetic landscape and gene expression. ChIP-seq enable the identification of histone marks that are regulated by ion, and thus impacted by ironomycin treatment. Most importantly, the most important outcome if the research is the discovery of a novel iron-endocytosis mechanism that prevails in mediating iron uptake in the persister cancer cell state (4 published in Nature Chemistry 2020).

These findings are a natural development of the grant proposal and have led to several new groundbreaking discoveries, namely: 1. Established general methodologies to visualize small molecules in cells (click imaging) and identify the genomic (drug-seq/chem-seq/click-seq) and non-genomic mechanistic targets of small molecules, 2. identified lysosomal iron and mitochondrial copper as druggable targets, 3. Identified copper and iron as regulators of metabolic and epigenetic programming of cell plasticity and the acquisition of the mesenchymal cancer cell state, discovered CD44 as main regulator of iron uptake.

This provides the opportunity to eradicate persister drug-tolerant cancer cells or block plasticity to re-sensitize cancer cells to standard of care.

toc.jpeg