Figure 5. 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 ± s.d. n = 3). The non-targeting 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 ± s.d. n = 3). (d) Quantification of AnnexinV-positive cells of murine NUP98/JARID1A-driven leukemia cells expressing indicated shRNAs after two rounds of replating (mean ± s.d. 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 ± s.d. n = 3). (g) Representative image of colonies from wild type or Cdk6^-/- NUP98/NSD1 leukemia cells. (h) Kaplan-Meier survival curves of mice transplanted with wild type vs. Cdk6 ^-/- fetal liver cells transformed with NUP98/NSD1 (n = 4). (i) Kaplan-Meier survival curves of secondary transplantations of bone-marrow-derived wild type or Cdk6 ^-/- leukemia cells at indicated concentrations into sublethally irradiated (4.5 Gray) recipient mice (5x10⁵ transplanted cells: n ≥ 3, 1x10⁵ transplanted cells: n = 5).