Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - Drug-Seq (Unravelling the Genomic Targets of Drugs Using High-Throughput Sequencing)

Teaser

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...

Summary

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.

Work performed

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. Marmycin
The natural product marmycin, an anthraquinone-derived natural product previously proposed to kill a variety of cancer cell lines through genome targeting. With the view to expand our repertoire of chemical tools suitable for cancer treatment and personalized medicine, we have developed the developed the first chemical synthesis of this anti-cancer natural product.

6. 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.

Final results

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 and APPOA as surrogates of cisplatin to visualize DNA-Pt in cells with high resolution. This staining was used to screen for other clinically approved drugs to evaluate their potential to sensitize genomes to cisplatin. We have 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 resitance mechanism into an apototic trigger. Therefore, this drug combination (e.g. Cisplatin/SAHA) can act synergistically at the level of chromatin. This is the first demonstration that targeting chromatin sensitize the genome to a genotoxic agent and is a major finding in the field (1 Paper submitted + 1 Patent).

2. 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).

3. Marmycin
Our work on Marmycin has led to the identification of a new mechanism of action for this class of natural products. Contrary to belief, we have made the important discovery that this fluorescent natural product does not target genomic DNA but operate instead as lysosomotropic small molecules, thereby leading to lysosomal membrane dysfunction and cancer cells death. This discovery opens up new opportunities for the treatment of cancers. We have elaborated on this finding to design a new drug named artesumycin that is 10 fold more potent (1 publication in Nature Chemistry).

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. This leads cellular iron depletion, which likely account for the indirect induction of DNA damage by these drugs. Most importantly, we have discovered that cancer stem cells up-regulate iron homeostasis (1 publication in Nature Chemistry). 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.