RIOK2 is required for maintaining AML in vivo. (A) Genetic locus and crossing strategy of the Riok2^tm1aKOMPWtsi mouse. After rederivation, the mice were crossed with FlpE mice to excise the LacZ+neo^R cassette. The progeny of this cross was then crossed with ROSA26::Cre^ERT mice to induce Cre-mediated excision of exons 4 and 5 of Riok2 after tamoxifen treatment. (B) Experimental outline of the Riok2^fl/fl R-Cre^ER MLL-AF9 cell line generation. c-Kit⁺ cells were isolated from a C57/BL6 carrying homozygous floxed Riok2 alleles and transduced with a retrovirus expressing the MLL-AF9 oncogene. After 5 rounds of replating, the cells were transplanted into sublethally irradiated mice and isolated after the mice developed full-blown leukemia. (C) Agarose gel showing PCR products from the amplified region of the floxed Riok2 locus after a 72-hour treatment of Riok2^fl/fl R-Cre^ER MLL-AF9 cells with EtOH or OHT (left). Western blot showing RIOK2 protein levels in Riok2^fl/fl R-Cre^ER MLL-AF9 cells 72 hours after addition of EtOH or OHT to the culture medium (right). (D) Growth curve of MA9 Riok2^fl/fl R-Cre^ER cells after OHT or EtOH addition to the culture medium (n = 3 biological replicates). Multiple unpaired Student t tests were performed to assess statistical significance between the OHT- and EtOH-treated cells. ***P < .001. Error bars represent standard deviation. (E) Experimental overview testing the role of Riok2 in vivo. Riok2^fl/fl R-Cre^ER MLL-AF9 cells were transplanted into sublethally irradiated B6/SJL mice. Fourteen days after transplantation, mice were injected with either tamoxifen or oil and monitored for survival. (F) Survival curve of 14 B6-SJL mice receiving transplants of with MA9 Riok2^fl/fl R-Cre^ER cells. Dotted lines indicate the time (days 14-19) when the mice were injected daily with either tamoxifen (n = 6) or oil (n = 8). A log-rank (Mantel-Cox) test was performed to assess differences in survival (P = .0003). (G) Spleen weights of mice at the time of death or experimental end point (day 74). Error bars represent standard deviation (SD). A Student t test was performed to assess significance. ****P < .0001.