Fluorogenic Ligand Enables Identification of NSD3-Overexpressed Tumors by Targeting the PWWP1 Domain of NSD3

Nuclear receptor-binding SET domain-containing 3 (NSD3) is a histone-modifying enzyme that is responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription. The human NSD3 is frequently amplified in a variety of cancers, including breast and squamous lung cancer. In addition to the methyltransferase domain (e.g., SET), NSD3 also harbors chromatin-reading modules, such as the proline-tryptophan-tryptophan-proline (PWWP) domain, which are required for the viability of cancer cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a fluorogenic ligand LY-06 by introducing an environment-sensitive fluorophore to the key structure of BI-9321 to detect and localize NSD3 in living cancer cells and tumors from xenograft mouse models. LY-06 enables targeting the PWWP1 domain of NSD3 with good in vitro binding affinity (<i>K</i>_d = 1.69 ± 0.05 μM), similar to that of BI-9321. Furthermore, both pharmacological and genetic studies (i.e., <i>NSD3-PWWP1</i> knockout) demonstrate that LY-06 specifically targets the methyl-lysine binding site of the PWWP1 domain in living cancer cells. Moreover, the application of LY-06 also enables distinguishing NSD3-overexpressed tumors from the wild type in xenograft mouse models. These results suggest that a strategically designed fluorogenic probe allows the assessment of NSD3 distribution in both living cancer cells and tumor tissues, thereby holding promise as a useful chemical tool to elucidate the underexplored biological functions of the PWWP1 domain of NSD3.