Development of chemical probes for NSD2 and its PHD fingers

1) Introduction

Epigenetics refers to mechanisms responsible for altering gene activity without changing the DNA sequence, which are essential for cell differentiation and physiological functions. Epigenetic modifications, such as histone post translational modifications (PTMs), play an important role in chromatin condensation and regulating gene expression, and an aberrant expression of epigenetic proteins are often associated with thedevelopment of various diseases, e.g. cancers and autoimmune disorders.

NSD2 belongs to the nuclear receptor-binding SET Domain (NSD) histone methyltransferase family and is involved in methylation of the side chain of lysine residues on histone H3 and H4, predominantly in dimethylation of H3K36 in vivo, showing effects on chromatin accessibility and gene expression of key regulators in apoptosis, DNA repair and cell adhesion. Historically, NSD2 is known for its link with Wolf-Hirschhorn Syndrome (WHS), where its hemizygous deletion of chromosomal region containing NSD2 gene leading to disorder of embryonic development and malformation syndrome. NSD2 overexpression is also found in several cancers (e.g. breast cancers, glioblastoma, prostate, lung, etc.) and it is associated with tumour aggressiveness and poor prognosis, but it especially seems to be crucial in the development of Multiple Myeloma (MM) and Acute Lymphoblastic Leukaemia (ALL). Specifically, MM cells with t(4;14) translocations show an aberrant expression of NSD2 leading to an increase of H3K36me2 levels, which is an active mark of gene expression, and a reduction of the repressive mark H3K27me3. This involves the development of altered cell growth and adhesion proprieties, which are lost after the knockdown of NSD2 isoforms using shRNA. Thus, inhibition of NSD2 may be considered a possible strategy for intervention in cancer, to develop new therapeutic approaches, and to understand the roles of NSD2.

Structurally, NSD2 is composed of a catalytic SET domain, an HMG (high mobility group) box, 2 PWWP (proline - tryptophan - tryptophan - proline) domains and 4 PHD (plant homeodomain) zinc fingers. Inhibition of NSD2 may be achieved by targeting the SET domain, or allosteric binding sites. Unfortunately, few small molecule inhibitors of NSD2 have been identified, e.g. N-Alkyl Sinefungin, LEM-06 and LEM-14, which show low selectivity and potency. The main problem for structure-based drug design with NSD2 is the difficulty in crystallizing the catalytic SET domain. In addition, the development of selective inhibitors a
gainst the SET domain is hampered by challenging assay development, with only nucleosome substrates known so far. To overcome some of these difficulties it may be beneficial to attempt new methods of targeting NSD2, such as using cyclic peptides or targeting its reader domains.

2) Aims of the project

The aims of this project are to develop cyclic peptides as chemical probes for NSD2, and to further the understanding of its PHD fingers in the regulation of NSD2 activity. Using cyclic peptides may be a promising approach in the development of chemical probes/inhibitors for 'undruggable' targets like NSD2, since they are able to interact with their targets over larger surface areas. Furthermore, targeting the reader domains of NSD2, especially its PHD fingers, may be an interesting alternative to targeting of the SET domain, since the reader domains can affect the recruitment of NSD2 to its target loci and its catalytic activity.