Figure 3. Smc3 haploinsufficiency... | Rockefeller University Press

Figure 3.

$Figure 3. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce AML. (A) Kaplan-Meier survival curve of primary transplant recipients of 106 total BM cells from Smc3fl/+ (n = 6), Smc3Δ/+ (n = 5), Flt3ITD (n = 5), and Smc3Δ/+ Flt3ITD mice (n = 5). (B) Kaplan-Meier survival curve of secondary and tertiary transplantation of BM from Smc3Δ/+ Flt3ITD mice (n = 5 each). (C) Peripheral blood counts of secondary transplanted mice with BM from Smc3fl/+ Flt3-ITD primary mice at the time of death (n = 5). (D) Mx1-cre–mediated deletion of one Smc3 allele reduces Smc3 expression in Smc3Δ/+ and Smc3Δ/+ Flt3ITD mice compared with age-matched controls (Smc3fl/+ and Flt3ITD). (E) Liver and spleen weights for primary mice (n = 4–8 for each genotype). (F and G) H&E stain of BM cytospin (F) and spleen (G). (H) Nucleolar stain of Flt3ITD and Smc3Δ/+ Flt3ITD BM. ACK-lysed BM was stained with TOTAL-NUCLEAR-ID fluorescent reagents, allowing simultaneous staining of both the nucleoli (green) and total nucleus (red). (I) H&E stain of MDS patient with refractory anemia with excess blasts-1 (RAEB-1) with molecular genetics identifying SMC3 K288fs and FLT3-ITD. Further experiments also identified IDH2 R140Q. Light microscopy at 100 magnification reveals multiple nucleoli in the blast population. (J) Serial plating and colony counts in methylcellulose for Smc3Δ/+ Flt3ITD and Flt3ITD BM. Whole BM was plated in triplicate at 20K cells/well. (K) Flow cytometric enumeration of BM LSK (Lin− Sca1+ c-Kit+). (L–O) enumeration of MPs (Lin− Sca1− c-Kit+), CMPs (lineage− c-Kit+ Sca-1− FcγR− CD34+), GMPs (lineage− c-Kit+ Sca-1− FcγR+ CD34+), and MEPs (lineage− c-Kit+ Sca-1− FcγR− CD34−; L) and flow cytometric enumeration of ST-HSCs (LSK Cd150+ Cd48+; M), LT-HSCs (LSK CD150+ CD48−; N), and MPP cells (LSK CD150− CD48+; O; n = 5–8 mice per genotype). Data are expressed as frequency of live cells per femur. Error bars represent ± SD (C, E, and J–O); *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test).$

Smc3 haploinsufficiency cooperated with Flt3-ITD to induce AML. (A) Kaplan-Meier survival curve of primary transplant recipients of 10⁶ total BM cells from Smc3^fl/+ (n = 6), Smc3^Δ/+ (n = 5), Flt3^ITD (n = 5), and Smc3^Δ/+Flt3^ITD mice (n = 5). (B) Kaplan-Meier survival curve of secondary and tertiary transplantation of BM from Smc3^Δ/+Flt3^ITD mice (n = 5 each). (C) Peripheral blood counts of secondary transplanted mice with BM from Smc3^fl/+Flt3-ITD primary mice at the time of death (n = 5). (D) Mx1-cre–mediated deletion of one Smc3 allele reduces Smc3 expression in Smc3^Δ/+ and Smc3^Δ/+Flt3^ITD mice compared with age-matched controls (Smc3^fl/+ and Flt3^ITD). (E) Liver and spleen weights for primary mice (n = 4–8 for each genotype). (F and G) H&E stain of BM cytospin (F) and spleen (G). (H) Nucleolar stain of Flt3^ITD and Smc3^Δ/+Flt3^ITD BM. ACK-lysed BM was stained with TOTAL-NUCLEAR-ID fluorescent reagents, allowing simultaneous staining of both the nucleoli (green) and total nucleus (red). (I) H&E stain of MDS patient with refractory anemia with excess blasts-1 (RAEB-1) with molecular genetics identifying SMC3 K288fs and FLT3-ITD. Further experiments also identified IDH2 R140Q. Light microscopy at 100 magnification reveals multiple nucleoli in the blast population. (J) Serial plating and colony counts in methylcellulose for Smc3^Δ/+Flt3^ITD and Flt3^ITD BM. Whole BM was plated in triplicate at 20K cells/well. (K) Flow cytometric enumeration of BM LSK (Lin⁻ Sca1⁺ c-Kit⁺). (L–O) enumeration of MPs (Lin⁻ Sca1⁻ c-Kit⁺), CMPs (lineage⁻ c-Kit⁺ Sca-1⁻ FcγR⁻ CD34⁺), GMPs (lineage⁻ c-Kit⁺ Sca-1⁻ FcγR⁺ CD34⁺), and MEPs (lineage⁻ c-Kit⁺ Sca-1⁻ FcγR⁻ CD34⁻; L) and flow cytometric enumeration of ST-HSCs (LSK Cd150⁺ Cd48⁺; M), LT-HSCs (LSK CD150⁺ CD48⁻; N), and MPP cells (LSK CD150⁻ CD48⁺; O; n = 5–8 mice per genotype). Data are expressed as frequency of live cells per femur. Error bars represent ± SD (C, E, and J–O); *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test).