The ATPase function of RIOK2 is essential for supporting leukemia proliferation. (A) Overview of the different RIOK2 mutants used for structure-function analysis. NES, nuclear export signal; wHTH, winged helix-turn-helix domain. (B) Competition-based proliferation assays in murine MLL-AF9 cells expressing the depicted RIOK2 mutants. Expression of luciferase (stuffer) was used as the negative control. MA9 cells were transduced with an sgRNA plasmid containing a BFP fluorophore to knock out endogenous Riok2. The percentage of positive cells was quantified by FACS and normalized to the nontargeting control relative to the initial time for each sgRNA. Data from a representative experiment are shown. (C) Growth curve of MA9 Riok2^fl/fl R-Cre^ER cells transduced with lentiviruses expressing the indicated proteins. OHT was added to the cell culture medium at day 0 (n = 3 biological replicates). Multiple unpaired Student t tests were performed to assess statistical significance comparing RIOK2 WT and RIOK2^{K123A, D246A}-expressing cell lines with the stuffer cell line. ***P < .001. ns, not significant. Error bars represent SD. (D) Diagram depicting RIOK2 phosphorylation sites based on data from PhosphoSitePlus.²⁵ Asterisks mark phosphorylation sites mutated in the RIOK2 4A (red asterisks) and 8A (red and black asterisks) mutants. (E) Competition-based proliferation assays in murine MLL-AF9 cells expressing WT RIOK2, 4A or 8A mutants as indicated. The cells were transduced with a lentivirus expressing BFP and the indicated Riok2 sgRNAs. The percentage of positive cells was quantified by FACS and normalized to the nontargeting control relative to the initial time point for each sgRNA. Data from a representative experiment are shown.