Figure 7. Hoxa9 and Meis1 overexpression rescues murine NHA9 driven leukemia from MLL1 dependence.

(A) Schematic for in vitro and in vivo Mll1 knockout rescue experiments.

(B) CFU assay of NHA9 in vitro transformed mouse BM LSKs, plated after 48 hr of 4-OHT treatments. Samples stating “+rescue” were infected with virus containing Hoxa9 and Meis1 and selected by hygromycin treatment, prior to excision. Bar graphs display mean number of colonies per 35 mm dish. Day 7 of the 1^st plating (left) and 2^nd plating (right). Data are representative of three individual experiments. (C) Shown is the mean percentage of blast versus non-blast colonies relative to all colonies counted per dish, per sample after the 1^st plating. (D) Representative images of colonies formed at day 7 of the 1^st plating. (E) Representative images of Wright-Giemsa–stained cytospin preparations of cells harvested at day 7 of the 1^st plating. (F) PCR analysis illustrates excision throughout the duration of a colony forming experiment. Representative gel images are shown. (G) Mouse BM LSKs were retrovirally transduced with NHA9 and Meis1a or both Meis1a and Hoxa9 (“+ rescue”). Sorted cells were injected into lethally irradiated C57Bl/6 mice. Plotted is the GFP⁺YFP⁺% of live cells in PB of mice before 4-OHT treatment and 14 days post treatment. A dot represents a single mouse in the experiment. (H) Survival curve. Mice were treated with 4-OHT at day 15 after transplant. (I) PCR analysis illustrates Mll1 excision in the murine BM of Cre-ER^T2;Mll1^f/f + Hoxa9/Meis1 recipient mice at time of death. Representative gel images are shown. See also Figure S7.

Abbreviations: Cre-ER^T2;Mll1^f/f + 4-OHT referred to as Mll1^−/−, Cre-ER^T2;Mll1^f/+ + 4-OHT referred to as Mll1^+/−, Mll1^f/f referred to as Mll1^+/+

* p < 0.05, ** p < 0.01. Error bars represent SD of mean.

^a.Log-rank test p value comparing survival for Cre-ER^T2;Mll1^f/f to Mll1^f/f

^b.Log-rank test p value comparing survival for Cre-ER^T2;Mll1^f/f to Cre-ER^T2;Mll1^f/f + Hoxa9/Meis1

^c.Log-rank test p value comparing survival for Cre-ER^T2;Mll1^f/f to Cre-ER^T2;Mll1^f/f + Hoxa9/Meis1 without 4OHT treatment