Frontiers in epigenetic chemical biology

Epigenetic events, somatically inherited through cell division, are potential drivers of acquired drug resistance in cancer. The high rate of epigenetic change in tumours generates diversity in gene expression patterns that can rapidly evolve through drug selection during treatment, leading to acquired resistance. Furthermore, persistent cell populations may survive drug exposure and have changes in gene expression associated with histone marks and chromatin modifying enzymes: these cells are reversibly drug tolerant, but can acquire resistance after further growth that becomes fixed by DNA methylation. Pharmacological reversion of repressive DNA and histone epigenetic marks can lead to re-sensitisation of tumours to chemotherapy in experimental models and some epigenetic drugs have entered clinical trials as resistance modulators with mixed success. Given the complexity and diversity of tumours, it is perhaps unrealistic to expect that drug resistance can ever be completely overcome. However, prevention of emergence of resistance by targeting epigenetically poised states, rather than reversal of a fixed epigenetic state, may be a more fruitful approach. Patient-derived xenograft models of acquired resistance, combined with gene targeted approaches such as CRISPR RNA-guided endonuclease Cas9, locus specific editing of histone modifications or chemical biology approaches using an epigenetic targeted pharmacopeia, that allow functional consequences of gene or mechanism specific intervention to be examined, has potential to provide innovation and insight into mechanisms of acquired resistance.

Epigenome-wide Association Studies have resulted in epigenetic mutations which might serve as diagnostic, prognostic, or even therapeutic targets. To functionally validate and further exploit such epigenetic biomarkers as therapeutic targets, epigenetic editing provides a powerful approach.

In epigenetic editing, epigenetic writers or erasers are fused to DNA binding platforms, such as engineered Zinc Finger Proteins (ZFPs) or the recently introduced CRISPR-Cas platform. CRISPR-Cas is based on designed RNA molecules which guide a nuclease (Cas9) to its target DNA site. Upon mutation of the nuclease activity (dCas), dCas can be fused to transcriptional effectors. Such Artificial Transcription Factors (ATFs) can modulate the expression of any gene at will, although the effects are presumed to be transient. To induce sustained gene expression modulation, we exploit these DNA binding platforms to target epigenetic effector domains to genomic loci of interest. 

Re-expression of a tumour suppressor gene generally is effective and induces apoptosis. Repression of gene expression can also easily be obtained. Guidelines on how to stably interfere with epigenetic gene regulation, however, are currently largely lacking. These data demonstrate feasibility of long-term gene repression as well as re-expression. Epigenetic Editing thus opens novel therapeutic avenues, realising the curable genome concept. 

The recent discovery of oxidized 5-methylcytosine (mC) derivatives in mammalian genomes and their involvement in key biological processes has created a need for methods that allow for their simple analysis at user-defined genomic positions. 

The group has recently started to explore the potential of transcription-activator-like effectors (TALEs) as programmable DNA-binding receptors for the analysis of 5mC and its derivatives. TALEs recognize DNA via the major groove that contains unique chemical information for each of the nucleobases and thus offer an ‘expanded programmability of DNA recognition’. Professor Summerer here reports on the engineering of new nucleobase selectivities into TALEs, and their use for epigenetic nucleobase analysis in genomes by affinity enrichment. These studies establish TALEs as the only receptor molecules capable of a direct isolation of user-defined epigenetic DNA biomarkers from genomes. 

It is now evident that there are numerous chemical modifications that occur naturally in DNA across species. These modifications can be incorporated and removed by natural enzymes and they can alter the structural and functional properties of the genome. Sir Shankar will discuss some recent studies that developed chemical methodologies to detect and decode modified bases in the genome and the applications of such methodologies to elucidate their roles in biology.

The protein methyltransferases (PMTs) constitute a large class of enzymes that catalyse the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example, gene amplification, chromosomal translocations and point mutations. The group is approaching drug discovery efforts against these enzymes in two distinct ways. First, they are approaching the PMTs as a target class, taking advantage of common aspects of their enzymology to develop a broad platform for small molecule inhibitor discovery. The second approach targets specific enzymes that are genetically altered in cancer, and for which strong evidence of an oncogenic role of enzymatic activity has been established. To date, the group has advanced small molecule inhibitors against three PMT targets to human clinical trials: pinometostat, an inhibitor of DOT1L for MLL-rearranged leukaemia; tazemetostat, an inhibitor of EZH2 for non-Hodgkin’s lymphoma, INI1-negative solid tumours and mesothelioma; and EPZ015938/GSK3326595, an inhibitor of PRMT5 for non-Hodgkin’s lymphoma and solid tumours.  Preclinical and clinical data for these investigational drugs will be presented.

Post-translational modifications of histones are closely associated with changes in transcription. Changes in histone lysine methyl marks are among the most prominent changes, and are mediated by methyltransferases and demethylases. Many diseases are characterised by transcriptional imbalances, and interference with epigenetic targets can be used to modulate these aberrant profiles. Here Dr Maes will report on the advances in the development of ORY-1001/RG6016, a potent selective inhibitor of LSD1, for oncology; and of ORY-2001, a dual inhibitor of LSD1 and MAO-B, for the treatment of neurodegenerative diseases. 

LSD1 inhibition compromises the leukaemic stem cell capacity in AML, and drives differentiation of blasts towards a more mature phenotype. Using a chemical probe for LSD1, it was initially shown that MLL translocated cells exhibit special sensitivity to LSD1 inhibition. Treating AML cells with the potent selective LSD1 inhibitor ORY-1001, the group confirmed responses at subnanomolar to nanomolar concentrations, and revealed that phenotypic changes were accompanied by a shift of the gene expression partially rebalancing the transcriptional profile towards that of normal monocytes/macrophages. The group has developed an activity based LSD1 chemoprobe and used it to pull down the protein and to unravel its network of interacting factors in AML. ORY-1001 has recently finalised a Phase I/IIa trial in relapsed or refractory acute leukaemia and these data corroborate the in vivo potency of ORY-1001 as an AML differentiating agent. LSD1 inhibitors are also highly active in SCLC, and ORY-1001/RG6016 is currently in a Phase I trial in this indication.

The potential role of LSD1 as a drug target is not limited to cancer. LSD1 is expressed in the brain and has a dual role in neuronal stem cell proliferation and neuronal differentiation / neurite extension. Using a proprietary chemoprobe based target engagement assay, the group has shown that they can modulate LSD1 activity in the brain using the brain penetrant dual inhibitor ORY-2001. Treatment with ORY-2001 in SAMP8 mice, a model for accelerated ageing and Alzheimer’s disease, rescues memory in the novel object recognition test. Again, it was found that phenotypic changes were accompanied by a shift and partial restoration of gene expression patterns in the hippocampus. A Phase I trial with ORY-2001 to assess the compounds’ tolerability, pharmacokinetics and pharmacodynamics in healthy young and elderly volunteers is nearing finalisation.

The family of bromodomain-containing proteins is an emerging class of epigenetic regulators that act as readers of the ‘histone code’ via recognition of proteins that have been specifically acetylated by histone acetyltransferases. Fuelled by the publication, in 2010, of high affinity inhibitors of the BET bromodomain subfamily of protein that have diverse therapeutic potential, this protein family has become an intensive area of research in industry and academia. Over 200 papers have been published since 2016, and BET compounds are now in clinical trials for oncology. However, despite this impressive demonstration of the potential of the BET subfamily as drug targets the function of other family members remain poorly understood.

To help to understand the potential of other bromodomains the group has developed chemical probes for use in cellular biology studies. Some properties of good chemical probes (potency, selectivity and cell permeability) may need to be equivalent to or even better than drugs, so their discovery can present significant challenges. This presentation will include the structural, computational and medicinal chemistry optimisation of fragment hits to chemical probes for the bromodomains of BRPF1 and ATAD2. How selectivity is assessed for multidomain complexes and the broader experience of using chemical probes for target validation will also be discussed.

NAD⁺-dependent histone deacetylases (sirtuins, SIRT1-7) have emerged as potential therapeutic targets for treatment of human illnesses such as cancer, metabolic, cardiovascular and neurodegenerative diseases, and other age-related disorders. In Professor Mai's lab, chemically different series of sirtuin inhibitors (SIRTi) and activators (SIRTa) have been identified so far.

Among SIRTi, some sirtinol analogues, obtained by replacement of the benzamide linkage of the prototype with other bioisosteric groups, have been described to induce apoptosis and/or cytodifferentiation in human leukaemia U937 cells. One of them, salermide, was well tolerated by mice and prompted tumour-specific apoptosis in a wide range of human cancer cell lines. In addition, the group designed some (thio)barbituric acid analogues (BDF4s) whose prototype, MC2141, displayed in U937 cells high apoptosis induction and showed antiproliferative effects against cancer cells including cancer stem cells. More recently, the gorup reported a series of highly specific SIRT2 inhibitors based on the 1,2,4-oxadiazole scaffold, inducing apoptosis and/or antiproliferative effects in leukaemia.

In contrast to the number of SIRTi, only few SIRT activators are known. A number of 1,4-dihydropyridines (DHPs) were described by us as SIRT1 activators able to increase nitric oxide levels in human keratynocyte HaCat cells, and to ameliorate skin repair in a mouse model of wound healing. In addition, the group identified, some pyrrolo[1,2-a]quinoxalines as the first synthetic SIRT6 activators. Biochemical assays show direct binding to the SIRT6 catalytic core and potent activation of SIRT6-dependent deacetylation. Crystal structures of SIRT6/activator complexes reveal that the compounds bind to a SIRT6-specific acyl channel pocket and identify key interactions. These results establish potent SIRT6 activation with small molecules and provide a structural basis for further development of SIRT6 activators as tools and therapeutics.