RIOK1 kinase activity is required for cell survival irrespective of MTAP status

Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers

Imaging by Atomic Force Microscopy of the Plasma Membrane of Prestin-Transfected Chinese Hamster Ovary Cells

The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distributed throughout the OHC plasma membrane. This protein has been named “prestin.” However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestin-transfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8–12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules