Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells - PubMed (original) (raw)

Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells

Min Huang et al. Oncotarget. 2016.

Abstract

The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.

Keywords: AML; BET inhibitors; HEXIM1; NPM1; autophagy.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declared that no conflicts of interest exists.

Figures

Figure 1

Figure 1. Proteolysis of NPM1 and HEXIM1 in AML cells expressing MLL-fusion proteins or NPMc+

(A) Western blot of whole cell lysates from leukemic and lymphoma cell lines. Cell lines: MV4-11 (MLL-AF4), ML2 (MLL-AF6), THP-1 (MLL-AF9), OCI-AML3 cells (NPMc+), K562, Raji, Karpas299, and SU-DHL-1 cells. C-Ab recognizes the C-terminus of wt-NPM1; N-Ab recognizes the N-terminus of both NPM1 and NPMc+. C (CF)- and N (NF)-terminal degradation fragments of NPM1/NPMc+ are indicated. (B, C) Western blots of lysates of leukemic blasts obtained from six NPMc+ AML patients with de novo disease and two MLL fusion AML patients. The characteristics of the primary AML cells are shown in Supplementary Figure 11.

Figure 2

Figure 2. Reduction of proteolytic degradation of NPM1/NPMc+, HEXIM1, and LC3B with autophagy inhibition

(A, C) OCI-AML3 and ML2 cells stably expressing inducible Atg5 shRNA were treated with vehicle or doxycycline for six days to induce Atg5 shRNA expression, followed by Western blot analysis. (B, D, E, F) OCI-AML3, MV4-11, ML2 cells, and OCI-AML3 cells stably expressing eGFP-LC3B were incubated with the autophagy inhibitors 3-MA, Wortmannin, or BafA1 at the concentrations shown for 20 h or 22 h followed by Western blot analysis. (G) Flow cytometric analysis of eGFP-LC3B fluorescent intensity in cells expressing eGFP-LC3B fusion protein after incubation with 3-MA (5 mM), wortmannin (10 μM), or BafA1 (50 nM, 100 nM) for 24 h. MFI is the mean fluorescent intensity of GFP-LC3B. Graphs represent the mean ± S.D. of biological triplicate. Asterisks (**) and (***) indicate p < 0.01 and p < 0.001, respectively, in relation to control un-treated cells.

Figure 3

Figure 3. Effects of BET inhibitors on protein expression and autophagy

(A, B, C) OCI-AML3 cells or primary NPMc+ AML cells from patient BM1 were treated with I-BET151 or JQ1 at the indicated concentrations for 24 h, followed by Western blot analysis of the proteins shown. (D, E) OCI-AML3 cells stably expressing eGFP-LC3B were treated with the BET inhibitors JQ1,I-BET151, 3-MA, or (−) JQ1 at the concentrations shown for 24 h, followed by flow cytometric analysis of GFP-LC3B fluorescent intensity (D) or Western Blot analysis (E). Bar graphs in Figure 3D represent the mean ± S.D. of biological triplicate. NS represents non-significance. Asterisks (***) indicate p < 0.001 in relation to control un-treated cells.

Figure 4

Figure 4. Effects of BET inhibitors on mRNA expression and the cellular distribution of NPM1/NPMc+ and HEXIM1 in OCI-AML3 cells

(A, B, C) Effects of JQ1 on the mRNA expression of NPM1, Bcl2, and HEXIM1 in OCI-AML3 cells. OCI-AML3 cells were untreated or treated with JQ1 at the concentrations shown for 24 h, followed by q-PCR analysis. The relative levels of mRNA expression were calculated using the 2−ΔΔCt method after normalization to the GAPDH level and were expressed as fold changes relative to control (set at 1). The mean ± S.D. of four replicates is represented in the bar graphs of Figure 4A, 4B, 4C. Asterisks (*) and (***) indicate p < 0.05 and p < 0.001, respectively, in relation to control cells. (D) Representative immuno-stained images of OCI-AML3 cells untreated or treated with JQ1 at the concentrations shown for 24 h. The N-terminal NPM1 antibody (N-Ab, green) recognizes both wt-NPM1 and NPMc+. The staining for DAPI was shown in blue. Scale bar, 200 px. (E) OCI-AML3 cells were untreated or treated with JQ1 at the indicated concentrations for 22 h, followed by cytoplasmic (column C) and nuclear (column N) fractionation and Western blot analysis.

Figure 5

Figure 5. Effects of autophagy inhibition on JQ1-induced apoptosis and protein expression in OCI-AML3 cells

(A) OCI-AML3 cells were untreated or treated with JQ1 at the indicated concentrations for 24 h or 48 h, followed by Western blot analysis of the proteins shown. Apoptosis Western Blot antibody Cocktail (pro/p17-caspase 3, cleaved-PARP, muscle actin) from Abcam (ab136812) detect apoptosis biomarkers caspase 3 and PARP, along with loading control muscle actin (42 kDa). The caspase 3 antibody detects both the 32 kDa pro-caspase 3 as well as the p17 subunit of active caspase 3 generated by cleavage of the pro-caspase 3 at Asp175. The PARP antibody detects only the apoptosis-specific 89 kDa PARP fragment (cleaved-PARP) generated from the full length PARP by active caspases. (B, C) Effect of Atg5-shRNA induction and the autophagy inhibitor 3-MA (5 mM) on JQ1-induced apoptosis in OCI-AML3 cells. (B) OCI-AML3 cells were treated with JQ1 or 3-MA alone or in combination at the indicated concentrations for 48 h, followed by flow cytometric analysis of apoptosis. Bar graphs represent the mean ± S.D. of biological triplicate. Asterisks (**) and (***) indicate p < 0.01 and p < 0.001, respectively, in relation to control cells. (C) OCI-AML3 cells stably expressing inducible Atg5 shRNA3 were incubated with doxycycline to induce Atg5 shRNA for five days, followed by treatment with JQ1 for 48 h and flow cytometric analysis of Annexin V positivity. Bar graphs represent the mean ± S.D. of biological triplicate. Asterisks (**) and (***) indicate p < 0.01 and p < 0.001, respectively, in relation to cells without doxycycline induction. (D) OCI-AML3 cells stably expressing inducible Atg5 shRNA3 were treated with vehicle or doxycycline to induce Atg5 shRNA for five days, followed by treatment with JQ1 at the indicated concentrations for 22 h, followed by Western blot analysis of the proteins shown.

Figure 6

Figure 6. Effects of autophagy inhibition on JQ1-induced apoptosis in primary NPMc+ AML cells

(A) Western blots of lysates of leukemic blasts obtained from five NPMc+ AML patients with relapsed disease. (B, C, D, E, and F). Viable mononuclear leukemic cells from each patient were treated with 3-MA (5 mM) or JQ1 alone or in combination at the indicated concentrations and times, followed by flow cytometric analysis of Annexin V positivity. Bar graphs represent mean values ± S.D. of biological triplicate. Asterisks (*), (**), and (***) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, in relation to cells without 3-MA treatment.

Figure 7

Figure 7. Effects of BET and autophagy inhibition on protein or mRNA expression and Annexin V positivity in MLL cell lines

(A, B) ML2 cells were treated with JQ1 at the indicated concentrations for 24 h, followed by Western blot analysis of the proteins shown or q-PCR analysis of HEXIM1, NPM1, and Bcl2. Bar graphs represent mean values ± S.D. of four replicates. Asterisks (*), (**), and (***) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, in relation to control cells. (C) ML2 cells were treated with JQ1 or 3-MA alone or in combination at the indicated concentrations and times, followed by flow cytometric analysis of Annexin V positivity. Bar graphs represent mean values ± S.D. of biological triplicate. Asterisks (**) and (***) indicate p < 0.01 and p < 0.001, respectively, in relation to cells without 3-MA treatment. (D) Effects of Atg5 depletion on JQ1-induced apoptosis in ML2 cells. ML2 cells stably expressing Atg5 shRNA3 were treated with vehicle or doxycycline to induce Atg5 shRNA for five days, then treated with JQ1 for 48 h, followed by flow cytometric analysis of apoptosis. The bar graphs represent mean values ± S.D. of biological triplicate. Asterisks (**) and (***) indicate p < 0.01 and p < 0.001, respectively, in relation to cells without doxycycline induction. (E) ML2 cells were treated with JQ1 or 3-MA (5 mM) alone or in combination at the concentrations shown for 48 h, followed by Western Blot analysis of proteins shown.

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