CDK6 is required for leukemia formation in vivo. (A) Experimental setup: Cdk6^+/+ and Cdk6^−/− BM cells were cocultivated on BCR-ABL^p210 producer cells and 2 × 10⁶ cells were injected i.v. in nonirradiated NSG mice. (B) Kaplan-Meier plot depicting disease onset of NSGs injected with Cdk6^+/+ or Cdk6^−/− BCR-ABL^p210+ leukemic cells (n = 6 and n = 7, respectively). Only 1 of 7 mice injected with Cdk6^−/− BCR-ABL^p210+ cells became diseased. All mice injected with Cdk6^+/+ BCR-ABL^p210+ cells became diseased within 3 months (***P < .001). (C) 2 × 10⁶ BM cells of diseased animals were transplanted in a second transplantation round and disease onset was monitored. None of the mice injected with Cdk6^−/− BCR-ABL^p210+ cells became diseased, but all mice injected with Cdk6^+/+ BCR-ABL^p210+ cells became diseased rapidly within 3 weeks (n = 5 and n = 6, respectively; ****P < .0001). (D) The experiment (Figure 6C) was terminated after 60 days and Cdk6^−/− nondiseased animals were euthanized. Contribution of Cdk6^−/− BCR-ABL^p210+–transformed cells (BM) was compared with diseased control animals at the time of terminal disease (n = 3 per genotype, ***P < .001). (E) Frequencies of BM BCR-ABL^p210+ LSKs of Cdk6^−/− animals compared with Cdk6^+/+ (diseased) animals. Cdk6^−/− BCR-ABL^p210+ LSKs were detectable in the BM (n = 3 per genotype).