Page 126 - Haematologica May 2020

C. Yu et al.
that autophagy may be a promising target pathway in BCR- ABL+ leukemia treatment.12-18 However, the distinct role of autophagy in the process of leukemogenesis is not com- pletely understood and crucial autophagy mediators have not been evaluated in in vivo leukemogenesis mouse mod- els.
BECLIN-1, a master regulator of autophagy, is essential for the formation of the autophagosome and autolysosome as a part of the Rubicon, VPS15, VPS34, ATG14, UVRAG and BECLIN-1 complex.19-23 An in vitro study has discovered that a treatment strategy combining TKI and inhibitors of BECLIN-1-mediated autophagy may be beneficial for BCR- ABL+ CML therapy,16 but in vivo data are missing and the molecular mechanisms underlying this effect remain unclear.
Methods
GST-pulldown assay, immunoprecipitation and Western blotting
All Beclin-1 fragments were cloned into PGEX-4T2 vector, which were confirmed by Sanger sequencing. Those con- structs were transformed into Bl21 competent cells, and a single clone was picked for culture in LB medium at 37°C with vigorous shaking. IPTG was added when the OD600 of the bacterial suspension reached 0.6. After an additional 2 hours (h) of incubation, bacterial cells were harvested, lysed using lysozyme and sonification and incubated for 3 h with glutathione-agarose beads in NETN buffer (0.5% NP40, 20 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Benzamidin, protease inhibitor cocktail [Roche]) at 4°C. The beads were then incubated with K562 cell lysates over night at 4°C. Immunoprecipitation and Western blotting were performed as described previously.24- 26 Briefly, immunoprecipitation was performed by adding IP lysis buffer (40 mM HEPES, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 50 mM NaF, 0.3% CHAPS, 1 mM sodium orthovanadate, 1 mM glycerolphosphate, protease inhibitor cocktail) to the cells for 1 h on ice. Pre-clearing of the lysates was performed using protein A or G agarose beads, followed by incubation with anti flag beads (Sigma) or antibody plus protein A or G beads (GE healthcare) overnight at 4°C. Protein extraction for Western blotting was performed using protein lysis buffer (10 mM Tris/HCl, 130 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 20 mM Na2HPO4/NaH2PO4, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 20 mM NaF, 1 mM glycerole 2- phosphate, protease inhibitor cocktail).
In vitro kinase assay
In vitro kinase assay was performed as described previous-
ly.27 Briefly, recombinant active ABL (ProQinase GmbH) was incubated with 10 mCi [γ-33P]ATP (PerkinElmer) and 1 mg recombinant GST-BECLIN-1 fragment in 50 mL kinase buffer (70 mM HEPES, 25 mM β-glycerophosphate, 3 mM MgCl2, 3 mM MnCl2, 1.2 mM DTT, 50 mg/mL PEG20.000, and 1% DMSO). Reactions were incubated at 30°C for 40 min. Proteins were separated by 10% SDS-PAGE, and phosphorylation was visualized by autoradiography.
Flow cytometry
Flow cytometric staining was performed as previously described.28-30
Mice
Mice were caged in special caging system with auto- claved food and acidified water at the University of Freiburg in accordance with national and institutional guidelines for animal care. All animal studies have been approved by the Ethics committees of the University Medical Center Freiburg and the district government in Freiburg (approval no. 35-9185.81/G-13/05).
Statistics
Statistical comparisons were performed using GraphPad Prism 6 software. Detailed statistical tests and significance cutoffs are indicated in each figure legend. All data repre- sent the mean ± standard error of the mean (SEM).
Study approval
The studies using human samples were conducted according to the Declaration of Helsinki principles. All biological samples were obtained following written informed consent from the patient and upon approval by the Ethics Committee of the University Medical Center Freiburg.
Additional methodology is provided in the Online Supplementary Materials and Methods.
Results
Knockdown of Beclin-1 delays BCR-ABL-mediated leukemogenesis in vivo
To further investigate the impact of autophagy in CML we examined the role of BECLIN-1, a master autophagy mediator, in BCR-ABL induced transformation and colony forming assays. Beclin-1 was downregulated using a tar- geted genetic approach with an individualized micro RNA-based knockdown of Beclin-1 in BCR-ABL-overex- pressing Ba/F3 cells and bone marrow derived cells (BMDC). Specific knockdown of Beclin-1 with two indi- vidually designed siRNA resulted in significantly lower proliferation of BCR-ABL transduced Ba/F3 cells com- pared to cells infected with a control miR sequence (Figure 1A-B). As the secondly designed Beclin-1 miR resulted in the most efficient Beclin-1 knockdown, we performed all further experiments solely with Beclin-1 miR2. We could detect higher apoptosis levels, but no decrease in cell cycle rate in Beclin-1 miR cells (Figure 1C, Online Supplementary Figure S1A ).
Furthermore, we could show significantly lower colony formation in BCR-ABL-expressing primary BMDC with Beclin-1 downregulating miR in comparison to control BMDC (Figure 1D-E).
Next, we examined the effects of Beclin-1 knockdown in a CML mouse model in vivo. BMDC from 5-FU pretreated animals were infected with a vector expressing BCR-ABL and the specific Beclin-1 (pMmiRBec–BCR-ABL) or control miR sequence (pMmiRCtrl–BCR-ABL) on one construct and under the LTR promoter. Survival of mice transplant- ed with BCR-ABL-expressing Beclin-1 knockdown BMDC was sustained and significantly prolonged compared to the control group (median survival 28 vs. 50 days, P<0.0001) (Figure 1F). Furthermore, the white blood cell count (WBC) and the leukemic burden of mice transplant- ed with Beclin-1 knockdown BCR-ABL BMDC was signif- icantly lower (87.3 vs. 14.8 x 103/mL on day 17, P<0.0001) compared to the control group (Figure 1G).
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