NUP98-fusion–protein-driven leukemia is dependent on CDK6 expression. (A) Western blot analysis of CDK6 levels in NUP98/JARID1A-expressing AML cells after 4 days of Dox-mediated Cdk6 shRNA induction. (B) Results of the competitive proliferation assay shown as the percentage of IRFP670-positive murine NUP98/JARID1A-driven leukemia cells expressing indicated shRNAs in the presence of Dox (0.5 µg/mL) over 13 days (mean ± SD; n = 3). The nontargeting shRNA (shRen.713) is used as neutral control, shRNAs targeting essential genes in hematopoietic cells Myb (shMyb.1653), or Myc (shMyc.1835) as positive controls. (C) Colony formation assay of G418-selected murine NUP98/JARID1A-driven leukemia cells expressing indicated shRNAs in the presence of Dox (0.5 µg/mL) over 4 rounds of replating. Colony numbers were normalized to cells expressing shRen.713 (mean ± SD; n = 3). (D) Quantification of AnnexinV-positive cells of murine NUP98/JARID1A-driven leukemia cells expressing indicated shRNAs after 2 rounds of replating (mean ± SD; n = 3). (E) Western blot analysis of CDK6 levels in wild-type vs Cdk6−/− fetal liver cells. (F) Colony formation assay of bone marrow–derived leukemia cells of mice transplanted with NUP98/NSD1-transformed Cdk6−/− or wild-type fetal liver cells. Colony numbers were normalized to wild-type NUP98/NSD1 leukemia cells (mean ± SD; n = 3). (G) Representative image of colonies from wild-type or Cdk6−/− NUP98/NSD1 leukemia cells, ×40 original magnification. (H) Kaplan-Meier survival curves with statistical analysis using the log-rank test of mice transplanted with wild-type vs Cdk6−/− fetal liver cells transformed with NUP98/NSD1 (n = 4). (I) Kaplan-Meier survival curves with statistical analyses using log-rank tests of secondary transplantations of bone marrow–derived wild-type or Cdk6−/− leukemia cells at indicated concentrations into sublethally irradiated (4.5 Gy) recipient mice (5 × 105 transplanted cells: n ≥ 3, 1 × 105 transplanted cells: n = 5). *P < .05, **P < .01, ***P < .001, ****P < .0001.