Figure 3. IKZF2 is highly expressed in LSCs and is required for maintaining LSC activity.

(A) Ikzf2 mRNA is highly expressed in LSCs, cells enriched in c-Kit^High cells compared to c-Kit^Low cells. Ikzf2 qPCR was performed in sorted c-Kit^High and c-Kit^Low cells from bone marrow of MLL-AF9 Ikzf2^f/f leukemic mice n=4, Student’s t test * p<0.05. (B) Representative flow plot showing the gating for c-Kit^High (top 15%) and c-Kit^Low (bottom 40%) cells, and also the gating for IKZF2 ^High cells shown in (C). (C) c-Kit^High cells have higher expression of IKZF2 compared to c-Kit^Low cells. Frequency of IKZF2^High population was measured in c-Kit^High and c-Kit^Low cells of MLL-AF9 leukemic mice. Mean and S.E.M of experiments from leukemic bone marrow cells isolated from Ikzf2^f/f n=15 mice ***, Student’s t test p<0.001.

(D) Colony Assay was performed with bone marrow leukemic cells from primary transplanted Ikzf2^f/f n=3 and Ikzf2^Δ/Δ n=3 mice. Student’s t test. *p<0.05. (E) Delay in survival between Ikzf2^f/f and Ikzf2^Δ/Δ mice is increased in secondary transplant of cells from (Fig 1D), compared to primary transplant. Result is from two combined transplants using four Ikzf2^f/f and five Ikzf2^Δ/Δ donors with recipients Ikzf2^f/f n=19 and Ikzf2^Δ/Δ n=26 mice. ***p<0.001. log-rank test. (F) Survival Curve of tertiary transplant of leukemic bone marrow cells from (E) shows worse LSC function when Ikzf2 is deleted. Transplant is shown with Ikzf2^f/f n=5 and Ikzf2^Δ/Δ n=5 mice ***p<0.001. log-rank test. (G) Survival Curve of quarternary transplant exhibits exhaustion of LSCs in Ikzf2^Δ/Δ mice. Three donors of each genotype from (F) were transplanted into recipients n=13 and n=15 mice for Ikzf2^f/f and Ikzf2^Δ/Δ, respectively. **p<0.01. log-rank test. (H) Ikzf2 deleted leukemic mice have reduced LSC frequency. Limiting dilution experiment assay performed with bone marrow cells from primary leukemic mice is shown. Left panel, table shows different number of cells and mice used for the transplant.. Right panel, graph showing the frequency of LSCs in Ikzf2^f/f and Ikzf2^Δ/Δ leukemic mice. ELDA software was used to calculate the frequency of the LSCs (Hu and Smyth, 2009). (I) Survival analysis for secondary transplant of the maintenance experiment is shown. Result is from transplanting Ikzf2^{f/f nonTAM}=10 and Ikzf2 ^{f/f prior TAM} n=10 mice, with two and one donors respectively. **p<0.01.