PO-346 The MYC antagonist MNT beyond MAX interaction

Introduction MNT has been described as an antagonist and modulator of MYC, one of the most prevalent oncoproteins in human cancer. Both MYC and MNT are bHLH-LZ transcription factors that heterodimerize with MAX, bind to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MNT) their transcription. Material and methods The cell lines used, URMT and URMax34, derive from MAX-deficient PC12 (rat pheochromocytoma), and carry a pHeBo-MT (empty vector) and a pHeBo-MT-MAX vector (MAX-inducible with Zn+2), respectively. Knockdown of MNT and MLX were achieved with short hairpin RNA constructs (shMNT and shMLX). Proliferation was assessed by cell counting and clonogenic assays; subG0-G1 population was determined by flow cytometry. RNA-seq was performed from two experiments of MNT silencing in URMT and URMax34 cells and confirmed by RT-qPCR. Changes in protein levels were analysed by western blot. Co-immunoprecipitation and proximity ligation assays were used to study protein-protein interactions. Results and discussions Knocking-down of MNT in UR61 cells resulted in an important decrease in cell proliferation, together with a decrease in both survivin and cyclin A, which are markers of pro-survival and cell proliferation, respectively. DNA content was measured by flow cytometry, revealing an increase in sub-G0 population in shMNT cells. Thus, MNT is required for optimal proliferation of these cells. This is the first evidence of a MAX-independent function of MNT. Then, we extracted RNA from two experiments of MNT silenced and carried out RNA-seq. This resulted in 158 genes whose expression was altered. Cell cycle, DNA replication and DNA repair genes were downregulated upon MNT silencing. However, there were other up-regulated genes like the cell cycle inhibitor CDKN1C (p57). As we confirmed gene regulation by MNT without MAX, we wondered whether it could be working as an heterodimer with MLX or as an homodimer. Co-immunoprecipitation and proximity-ligation assays showed MNT’s ability to form homodimers and heterodimers with MLX. Finally, we carried out MLX knockdown and determined the genes regulated by MNT-MLX or MNT-MNT complexes. Conclusion In summary, we report novel MAX-independent functions of MNT. In our MAX-deficient model, MNT can be found in homodimers (MNT-MNT) or heterodimers (MNT-MLX) and it supports proliferation and regulates cell cycle and DNA repair genes. This new data about MNT can open new insights into cell biology and tumour development promoted by MYC.