Figure 6.
Pharmacological inhibition of RIOK2 has antileukemic effects in vivo and in vitro. (A) Chemical structure of RIOK2i (1-[2-(2-thiazolyl)diazenyl]-2-naphthalenol). (B) Viability of MA9 andTrp53−/− MEFs, after 72 hours of incubation with RIOK2i at the indicated concentrations. The data points show the mean ± SD of 3 technical replicates; 50% effective concentrations (EC50) were calculated with GraphPad Prism. (C) Viability of BJ-hTERT, MOLM13, and THP-1 cells after 72 hours of incubation with RIOK2i at the indicated concentrations. The data points show the mean ± SD of 3 technical replicates. EC50 values were calculated with GraphPad Prism. (D) Growth curves of MA9 cells treated as indicated over the course of 4 days. The data points show the mean ± SD. Multiple unpaired Student t tests were performed to assess the statistical significance of the comparison of RIOK2i treatment conditions with the DMSO-treated condition. ***P < .001. The experiment was performed in 3 biological replicates. (E) OP-puro labeling of MLL-AF9 (left) and MOLM13 cells (right) treated as indicated for 48 hours. A Student t test was used to assess statistical significance. *P < .05; **P < .01. n = 3 technical replicates. (F) Quantification of FACS-based EdU labeling and estimation of cell cycle profiles of murine MA9 or MOLM13 cells treated with DMSO or 300 nM or 1 µM RIOK2i for 72 hours. Error bars represent standard error of the mean. (G) Experimental overview of RIOK2i in vivo treatment strategy. SJL-B6 mice were sublethally irradiated and injected with 50 000 GFP-MA9 Cas9 cells the following day. FACS analysis to validate engraftment of leukemic cells was performed after 14 days. After validation, mice were treated with RIOK2i for 3 days per week at a concentration of 120 mg/kg. FACS analysis was performed at days 21 and 25 to check for the presence of leukemic cells. (H) FACS-based quantification of GFP-positive leukemic cells in erythrocyte depleted peripheral blood (PB) comparing oil-treated with RIOK2i-treated mice at the indicated time points. The Student t test was used to assess statistical significance. *P < .05. ns, not significant. (I) Model explaining the functional consequences of RIOK2 loss on protein synthesis and ribosomal stability in leukemic cells.

Pharmacological inhibition of RIOK2 has antileukemic effects in vivo and in vitro. (A) Chemical structure of RIOK2i (1-[2-(2-thiazolyl)diazenyl]-2-naphthalenol). (B) Viability of MA9 andTrp53−/− MEFs, after 72 hours of incubation with RIOK2i at the indicated concentrations. The data points show the mean ± SD of 3 technical replicates; 50% effective concentrations (EC50) were calculated with GraphPad Prism. (C) Viability of BJ-hTERT, MOLM13, and THP-1 cells after 72 hours of incubation with RIOK2i at the indicated concentrations. The data points show the mean ± SD of 3 technical replicates. EC50 values were calculated with GraphPad Prism. (D) Growth curves of MA9 cells treated as indicated over the course of 4 days. The data points show the mean ± SD. Multiple unpaired Student t tests were performed to assess the statistical significance of the comparison of RIOK2i treatment conditions with the DMSO-treated condition. ***P < .001. The experiment was performed in 3 biological replicates. (E) OP-puro labeling of MLL-AF9 (left) and MOLM13 cells (right) treated as indicated for 48 hours. A Student t test was used to assess statistical significance. *P < .05; **P < .01. n = 3 technical replicates. (F) Quantification of FACS-based EdU labeling and estimation of cell cycle profiles of murine MA9 or MOLM13 cells treated with DMSO or 300 nM or 1 µM RIOK2i for 72 hours. Error bars represent standard error of the mean. (G) Experimental overview of RIOK2i in vivo treatment strategy. SJL-B6 mice were sublethally irradiated and injected with 50 000 GFP-MA9 Cas9 cells the following day. FACS analysis to validate engraftment of leukemic cells was performed after 14 days. After validation, mice were treated with RIOK2i for 3 days per week at a concentration of 120 mg/kg. FACS analysis was performed at days 21 and 25 to check for the presence of leukemic cells. (H) FACS-based quantification of GFP-positive leukemic cells in erythrocyte depleted peripheral blood (PB) comparing oil-treated with RIOK2i-treated mice at the indicated time points. The Student t test was used to assess statistical significance. *P < .05. ns, not significant. (I) Model explaining the functional consequences of RIOK2 loss on protein synthesis and ribosomal stability in leukemic cells.

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