Abstract 4357: Key differences revealed in NSD2 kinetics using truncated versus full-length protein
Authors: Pappalardi Melissa B. , Schneck Jessica L. , Matico Rosalie , Huddleston Michael , Hou Wangfang , McDevitt Patrick , Annan Roland , Kirkpatrick Robert , Kruger Ryan 
Published: August 2015

Histone lysine methyltransferases (HKMTs) are a class of enzymes that transfer a methyl group from S-adenosyl-L-methionine (SAM) to lysine residues on histone tails. The nuclear receptor SET domain-containing (NSD) family of proteins is known to methylate lysine 36 of histone H3 (H3K36). Abnormal methylation at H3K36 has been widely implicated in a variety of cancers and diseases, therefore, the enzymes responsible for this posttranslational modification are of interest from a drug-discovery standpoint. NSD2 (MMSET/WHSC1), a representative of this family, was found to be highly expressed in a multitude of human tumors and has been directly linked to multiple myeloma and Wolf-Hirschhorn syndrome. The chromosomal translocation t(4:14)(p16;q32) that occurs in 15% of myeloma patients is associated with both increased production of NSD2 and poor prognosis while loss of the NSD2 gene at the 4p16.3 region results in Wolf-Hirschhorn syndrome. In order to better understand the multi-dimensional nature of NSD2, full-length and truncated versions of the protein were generated to evaluate NSD2 kinetics. The biochemical activity of each NSD2 construct was assessed using (1) a radioactive assay measuring 3H transfer from SAM to the histone substrate or (2) LC-MS/MS analysis of the NSD2-dependent product. Full-length NSD2 prefers a nucleosomal substrate; whereas, C-terminal truncation of a highly charged region (AA 12-14-1240) resulted in a loss of nucleosomal activity and a gain of activity using a peptide derived from Histone H3. Additionally, LC-MS/MS mapping revealed a shift in the methylation site from H3K36 to H3K18 when using the truncated system. More detailed kinetic analysis revealed that the FL/nucleosome reaction catalyzes processive methylation while truncated NSD2 methylates the peptide distributively. Lastly, key changes in inhibitor specificity were observed. An alternative C terminal region, residues 1341-1365, was required to maintain potency of the product inhibitor SAH but not the close analog sinefungin. This may be indicative of the ping-pong kinetics proposed for FL NSD2 implying that SAH targets the NSD2-nucleosome bound form of the enzyme (UC vs. nucleosome) while sinefungin does not (NC vs. nucleosome). While a truncated NSD2/peptide system would be much easier to screen, profile and characterize in the search for inhibitors, the results found herein indicate that screening a full-length NSD2/nucleosome system may be more physiologically relevant.

Citation Format: Melissa B. Pappalardi, Jessica L. Schneck, Rosalie Matico, Michael Huddleston, Wangfang Hou, Patrick McDevitt, Roland Annan, Robert Kirkpatrick, Ryan Kruger. Key differences revealed in NSD2 kinetics using truncated versus full-length protein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2015-4357