Multi-omics analyses of MEN1 missense mutations identify disruption of menin–MLL and menin–JunD interactions as critical requirements for molecular pathogenicity

Abstract

Background Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. Results Here, we applied and in silico screening approach for 253 disease-related MEN1 missense mutations in order to select a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/MLL2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the MLL1 and MLL2 interactions when compared with JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. Conclusions Our results underline the effects of MEN1 gene mutations in both familial and sporadic tumors of endocrine origin on the interactions of menin with the MLL1 and MLL2 histone H3K4 methyltransferase complexes and with JunD-containing transcription factors. Menin binding pocket mutants R52G, E255K and E359K have differential effects on MLL1/MLL2 and JunD interactions, which translate into differential genomic binding patterns. Our findings encourage future studies addressing the pathophysiological relevance of the separate MLL1/MLL2- and JunD-dependent functions of menin mutants in MEN1 disease model systems.

Clinically defined mutations inMEN1alter its tumor-suppressive function through increased menin turnover

“…Prior studies focused on mapping structural defects at the protein level have shown that mutations arising in specific functional domains of menin can impair protein-protein interactions that may result in altered gene expression leading to cell proliferation, altered cell-specification, and NET development [24,[39][40][41][42] . For instance, a recent in silico analysis of 253 disease related MEN1 missense mutations identified three single nucleotide variants that impaired the binding affinity of menin with the Mixed Lineage Leukemia 1 (MLL) protein [42] . In contrast, these mutations did not have an apparent effect on the menin-JUND interaction, further raising the potential for context-specific regulation that might dictate the tumor suppressive and oncogenic properties of menin.…”

MENIN‐mediated regulation of gastrin gene expression and its role in gastrinoma development

“…MENIN acts as a tumor suppressor in NENs; 11 however, its exact mechanism of action is unclear. In contrast, MENIN functions as a co‐oncogene in some hematopoietic malignancies by directing a mutated lysine methyl transferase KMT2A/MLL protein to genes controlling cell proliferation 32,33 …”

“…Missense mutations have been identified throughout the whole coding domain, yet no hotspots or clear genotype–phenotype correlations have been predictably identified 38,40,41,42 . Despite this, the majority of MEN1 ‐associated missense mutations yield aberrantly functioning proteins that are unstable and exhibit reduced or absent nuclear MENIN levels compared to the wild‐type protein 32,43,44 . Nevertheless, more research is necessary to establish whether residual MENIN in tumors display a wild type or mutated genotype and phenotype.…”

“…38,40,41,42 Despite this, the majority of MEN1-associated missense mutations yield aberrantly functioning proteins that are unstable and exhibit reduced or absent nuclear MENIN levels compared to the wild-type protein. 32,43,44 Nevertheless, more research is necessary to establish whether residual MENIN in tumors display a wild type or mutated genotype and phenotype. In this instance, single-cell proteomics to assess the status of MENIN protein will be beneficial.…”

“…52 Analysis of specific MENIN missense mutations revealed that mutations in the MENIN interacting domain disrupts the function of JUND. 32 Taken together, these T A B L E 1 MEN1 functional domains, the amino acid positions that correspond to them on MENIN and MENIN-interacting proteins, and the roles that they play with a normal MENIN protein.…”