Abstract: Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here we present a recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX) in three individuals with overlapping macrocephaly-associated phenotypes. The variant, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription, not stabilization of CCND2 as observed for other macrocephaly-associated mutations in PI3K-AKT pathway components. We show that the purified b-HLH-LZ domain of MAX-Arg60Gln binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation.
To model the role of MAX during neurodevelopment, we used CRISPR/Cas9 to introduce the p.Arg60Gln variant into healthy iPSCs and used these to generate iPSC-derived neural progenitor cells (NPCs). We found a reduction in the neuronal marker β-tubulin (TUJ1) and increased expression of the NPC marker TBR2, compared to wildtype, indicating impaired or stalled differentiation of these neuronal precursors. Kinome activity profiling revealed significant upregulation of hepatocyte growth factor receptor (MET) kinase activity in MAX-Arg60Gln NPCs, as well as other receptor tyrosine kinases, such as PDGFRA, linked to cell cycle dysregulation and abnormal cell growth. Of interest, a recurrent phenotype in patients with the MAX R60Q variants is renal agenesis, with previous studies showing a crucial role for MET in kidney formation. Further work to model this mutation in kidney organoids is ongoing. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAX-Arg60Gln individuals.
