Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

Abstract

NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 +/- 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer

“…Indeed, efforts have already been made to develop an inhibitor of PHIP BD2 function ( Cox et al 2016 ). Recent studies have demonstrated that targeting a single reader domain in a multivalent chromatin protein can be sufficient to disrupt normal localization ( Dilworth et al 2021 ). Thus, our characterization of the substrate specificity of each reader domain in PHIP may open up additional approaches to target its function.…”

Lysine methyltransferase inhibitors: where we are now

“…Further structure-based optimization resulted in 103 (UNC6934), that showed a potent binding to PWWP1 (K D of 91 nM). 268 In order to better understand the binding mode of 103, a representation of the main interaction has been reported in Figure 29. The crystal structure of the inhibitor in complex with NSD2-PWWP1 (PDB 6XCG) shows how the cyclopropyl is inserted in a hydrophobic pocket made of Val230 and Phe266.…”

Targeting the Nuclear Receptor-Binding SET Domain Family of Histone Lysine Methyltransferases for Cancer Therapy: Recent Progress and Perspectives

“…Another recent study by the same group reported UNC6934 ( 34 ) as a first-in-class potent chemical probe that inhibits the interaction of NSD2-PWWP1 with the H3K36me2 nucleosome by selectively binding to the aromatic cage of the PWWP1 domain of NSD2 methyltransferase. Using the crystallographic information on NSD2 in complex with compound 32 and further molecular docking simulations, the group identified compound 33 (MRT866) containing a benzoxazinone bicyclic ring ( K d = 349 nM).…”

“…Using virtual screening and biophysical assays initially, they identified a chiral compound, 29, which showed selective interaction with the PWWP1 domain of NSD2 (K d = 41 μM). Based on the initial data on 29, the group designed a series of compounds using a ligand-guided scaffold hopping to find an improved active Another recent study by the same group 98 reported UNC6934 (34) as a first-in-class potent chemical probe that inhibits the interaction of NSD2-PWWP1 with the H3K36me2 nucleosome by selectively binding to the aromatic cage of the PWWP1 domain of NSD2 methyltransferase. Using the crystallographic information on NSD2 in complex with compound 32 and further molecular docking simulations, the group identified compound 33 (MRT866) containing a benzoxazinone bicyclic ring (K d = 349 nM).…”