Page 127 - Haematologica May 2020

BCR-ABL suppresses autophagy via BECLIN-1
Immunoblotting of splenocyte extracts of transplanted mice confirmed efficient downregulation of Beclin-1 one month after transplantation (Figure 1H). Upon disease induction, fluorescence-activated cell sorting (FACS) analyses of transplanted animals showed no differences in the immune phenotype of the BCR-ABL induced disease
by Beclin-1 downregulation (Online Supplementary Figure S1B-D). To test whether the impact of Beclin-1 knock- down on CML cells is due to a general effect of autophagy inhibition or more due to a specific role of BECLIN-1 in BCR-ABL induced diseases, we also deleted another main autophagy regulator, ATG5 in a CML mouse model: Atg5
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Figure 1. BECLIN-1 downregulation delays BCR-ABL-mediated proliferation in vitro and in vivo. (A) Immunoblot analyses were used to confirm downregulation of BECLIN-1 using two different Beclin-1 directed miR in Ba/F3 cells and BMDC. (B) Cell proliferation measurement was performed by MTT assay in Ba/F3 cells infected with indicated construct towards IL-3 withdrawal, indicating that BCR-ABL-mediated cell proliferation is impaired by Beclin-1 knockdown. ***P<0.001, *P<0.05, Student's t-test. (C) Statistical analysis of flow cytometric staining showing Annexin-V–/ propidium iodide (PI)- ("alive"), Annexin-V+ / PI–("apoptotic") and Annexin-V+ / PI+ ("dead") Ba/F3 cells with indicated construct. **P<0.01, *P<0.05, Student's t test. (D) Methylcellulose (MC) colony formation assay of primary 5-FU enriched bone marrow cells showed impaired colony formation upon BCR-ABL expression in Beclin-1 knockdown cells compared to control miR expressing cells. 1,000 EGFP+ BMDC infected with the indicated construct were plated into methylcellulose in the absence of growth factors and colonies were quantified 10 days later. One rep- resentative well is shown. Three independent experiments were performed in doublets. Grid size is 5 x 5 mm. (E) Quantitation of the MC shown in (D) (72 vs. 55.7 colony-forming unit [CFU], *P<0.05, and 72 vs. 41.7 CFU, **P<0.01, respectively, student’s t-test). (F) Kaplan-Meier curve demonstrates a significantly prolonged survival of mice transplanted with Beclin-1 knockdown BCR-ABL+ BMDC compared to control mice (Median survival 28 vs. 50 days, ***P<0.001 in two independent transplantations, Log-rank test (n=11, control miR; n=13, Beclin-1 miR)). (G) WBC from peripheral blood (PB) showed a significant reduction of leukemic progression in mice transplanted with Beclin-1 knockdown cells (11.6 vs. 4 million/mL, day 14, *P<0.05; 87.3 vs. 14.8 million/mL, day 17, ***P<0.001; 151.1 vs. 81.9 mil- lion/mL, day 21, *P<0.05). (H) Efficient and durable knockdown of Beclin-1 was proven by immunoblot analyses of spleen cells of transplanted mice (day 27).
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