American Society of Hematology

Figure 3.

$Figure 3. Loss of 1 copy of Ctnnb1 in an Apc-haploinsufficient microenvironment prevents or delays the development of MDS by 8 to 10 months. (A) Kaplan-Meier survival curves for Apcdel/+ (A; n = 4), Apcdel/+, Ctnnb1del/+ (AC; n = 11), Ctnnb1del/+ (C; n = 3), and Apcfl/+, Ctnnb1fl/+ (Cre−; n = 9) recipient mice. Median survival of Apcdel/+ and Apcdel/+, Ctnnb1del/+ recipient mice was significantly different (115 vs 413 days; P < .0001). All control mice (C and Cre−) survived until the end of the study, with the exception of 1 Apcfl/+, Ctnnb1fl/+ recipient that died at 343 days, likely due to a hemorrhagic renal cyst. (B) Percentage of CD71+Ter119+ erythroid cells, Gr1+CD11b+ myeloid cells, and CD19+IgM+ B cells in spleen isolated from Cre− (∼400 days), A (70-115 days), and AC (303-413 days) recipients that eventually displayed a fatal anemia. At sacrifice, the AC cell populations were more similar to A than Cre− recipients. (C) RBC and Hb counts in all 4 cohorts over time. The development of anemia is delayed in AC recipients after 35 weeks (a point in time when all A recipients have already been sacrificed due to severe anemia). In Cre− control recipients, 2 mice developed moderate anemia at 57 weeks. However, 1 mouse had an apparent colorectal tumor, and the other had a hemorrhagic renal cyst; neither had developed MDS. (D) MSCs were isolated from Cre− (control), A, and AC littermates 2 months posttreatment with pIpC to induce Cre-mediated deletion, and before development of disease. Following in vitro Wnt3a stimulation for 6 hours, nuclear and cytoplasmic fractions were isolated and immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) antibodies. Quantification of 3 independent experiments shows increased nuclear and cytoplasmic Ctnnb1 protein expression in Apcdel/+ MSCs that is reduced by ∼50% upon haploinsufficient loss of Ctnnb1. (E) RNA was isolated from Cre−, A, and AC MSCs (no Wnt3a stimulation), transcribed to complementary DNA (cDNA), and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ***P < .0001, **P < .001, *P < .05. NS, not significant.$

Loss of 1 copy of Ctnnb1 in an Apc-haploinsufficient microenvironment prevents or delays the development of MDS by 8 to 10 months. (A) Kaplan-Meier survival curves for Apc^del/+ (A; n = 4), Apc^del/+, Ctnnb1^del/+ (AC; n = 11), Ctnnb1^del/+ (C; n = 3), and Apc^fl/+, Ctnnb1^fl/+ (Cre⁻; n = 9) recipient mice. Median survival of Apc^del/+ and Apc^del/+, Ctnnb1^del/+ recipient mice was significantly different (115 vs 413 days; P < .0001). All control mice (C and Cre⁻) survived until the end of the study, with the exception of 1 Apc^fl/+, Ctnnb1^fl/+ recipient that died at 343 days, likely due to a hemorrhagic renal cyst. (B) Percentage of CD71⁺Ter119⁺ erythroid cells, Gr1⁺CD11b⁺ myeloid cells, and CD19⁺IgM⁺ B cells in spleen isolated from Cre⁻ (∼400 days), A (70-115 days), and AC (303-413 days) recipients that eventually displayed a fatal anemia. At sacrifice, the AC cell populations were more similar to A than Cre⁻ recipients. (C) RBC and Hb counts in all 4 cohorts over time. The development of anemia is delayed in AC recipients after 35 weeks (a point in time when all A recipients have already been sacrificed due to severe anemia). In Cre⁻ control recipients, 2 mice developed moderate anemia at 57 weeks. However, 1 mouse had an apparent colorectal tumor, and the other had a hemorrhagic renal cyst; neither had developed MDS. (D) MSCs were isolated from Cre⁻ (control), A, and AC littermates 2 months posttreatment with pIpC to induce Cre-mediated deletion, and before development of disease. Following in vitro Wnt3a stimulation for 6 hours, nuclear and cytoplasmic fractions were isolated and immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) antibodies. Quantification of 3 independent experiments shows increased nuclear and cytoplasmic Ctnnb1 protein expression in Apc^del/+ MSCs that is reduced by ∼50% upon haploinsufficient loss of Ctnnb1. (E) RNA was isolated from Cre⁻, A, and AC MSCs (no Wnt3a stimulation), transcribed to complementary DNA (cDNA), and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ***P < .0001, **P < .001, *P < .05. NS, not significant.

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