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. 2014 Oct 23;124(17):2687-97.
doi: 10.1182/blood-2014-03-564534. Epub 2014 Sep 10.

A Bim-targeting strategy overcomes adaptive bortezomib resistance in myeloma through a novel link between autophagy and apoptosis (V体育平台登录)

Shuang Chen  1 , Yu Zhang  2 , Liang Zhou  1 , Yun Leng  3 , Hui Lin  1 , Maciej Kmieciak  1 , Xin-Yan Pei  1 , Richard Jones  4 , Robert Z Orlowski  4 , Yun Dai  1 , Steven Grant  5
Affiliations

V体育2025版 - A Bim-targeting strategy overcomes adaptive bortezomib resistance in myeloma through a novel link between autophagy and apoptosis

Shuang Chen et al. Blood. .

V体育安卓版 - Abstract

Bim contributes to resistance to various standard and novel agents. Here we demonstrate that Bim plays a functional role in bortezomib resistance in multiple myeloma (MM) cells and that targeting Bim by combining histone deacetylase inhibitors (HDACIs) with BH3 mimetics (eg, ABT-737) overcomes bortezomib resistance. BH3-only protein profiling revealed high Bim levels (Bim(hi)) in most MM cell lines and primary CD138(+) MM samples. Whereas short hairpin RNA Bim knockdown conferred bortezomib resistance in Bim(hi) cells, adaptive bortezomib-resistant cells displayed marked Bim downregulation. HDACI upregulated Bim and, when combined with ABT-737, which released Bim from Bcl-2/Bcl-xL, potently killed bortezomib-resistant cells VSports手机版. These events were correlated with Bim-associated autophagy attenuation, whereas Bim knockdown sharply increased autophagy in Bim(hi) cells. In Bim(low) cells, autophagy disruption by chloroquine (CQ) was required for HDACI/ABT-737 to induce Bim expression and lethality. CQ also further enhanced HDACI/ABT-737 lethality in bortezomib-resistant cells. Finally, HDACI failed to diminish autophagy or potentiate ABT-737-induced apoptosis in bim(-/-) mouse embryonic fibroblasts. Thus, Bim deficiency represents a novel mechanism of adaptive bortezomib resistance in MM cells, and Bim-targeting strategies combining HDACIs (which upregulate Bim) and BH3 mimetics (which unleash Bim from antiapoptotic proteins) overcomes such resistance, in part by disabling cytoprotective autophagy. .

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Figures

Figure 1
Figure 1
Loss of Bim is associated with adaptive bortezomib resistance in Bimhi MM cells. (A) Immunoblotting analysis was performed to profile basal levels of the BH3-only proteins Bim (including EL, L, and S isoforms), Noxa, and/or Puma in untreated human MM cell lines. *Indicates nonspecific bands. (B) Three primary samples obtained from 2 patients with MM as well as normal CD34+ cells isolated from cord blood (CB) were subjected to immunoblotting analysis for basal levels of Bim. Blots for BimEL were quantified by using ImageJ software (available online). Values indicate fold-increase after normalization to loading controls (eg, glyceraldehyde-3-phosphate dehydrogenase [GAPDH] or β-actin [β-act]). (C) Bimhi U266 cells were stably transfected with constructs encoding shRNA-targeting Bim (shBim) or scrambled sequence as a negative control (shNC). Cells were then exposed to 5 nM bortezomib (Btz) for 24 hours, followed by immunoblotting for expression of Bim and cleavage of PARP (left panels) or flow cytometry to determine the percentage of apoptotic (annexin V+) cells (right panel). (D) U266 cells were continuously cultured with gradually increasing concentrations of bortezomib up to 15 nM to generate a subline (PS-R) that acquired marked resistance to bortezomib, as determined by flow cytometry (left panel). A Human Apoptosis RT2 Profiler Array Kit was then used to compare differences in the expression (messenger RNA [mRNA]) of key genes involved in apoptosis between PS-R and U266 cells. The heat map showed genes that were up- or downregulated in PS-R cells compared with U266 cells (right panel). (E) In parallel, immunoblotting analysis was conducted to monitor protein levels of Bim and/or Mcl-1 in untreated parental U266 cells and their bortezomib-resistant counterparts (PS-R). (F) Primary CD138+ cells were isolated from newly diagnosed (Dx; n = 2) or relapsed (R; n = 3, who had received prior bortezomib) MM patients, after which untreated cells were subjected to immunoblotting analysis (left) or immunofluorescence staining for CD138 (phycoerythrin, red) or Bim (AlexaFluor 488, green). Arrows and arrowheads indicate CD138+ cells displaying low and high Bim positivity, respectively. Scale bar = 10 μm; magnification ×40. α-tub, alpha tubulin; CF, cleaved fragment; DAPI, 4′,6 diamidino-2-phenylindole; L.E., long exposure, Veh, vehicle.
Figure 2
Figure 2
Bim acts as a determinant of ABT-737 sensitivity in Bimhi MM cells. (A) U266 (upper left), RPMI8226 (upper right), and MM.1S (lower) were treated with the indicated concentrations (nM) of ABT-737 for 24 or 48 hours, after which expression of Bim and Mcl-1 was monitored by immunoblotting analysis. (B) U266 cells stably transfected with Bim shRNA (left upper panels) were treated with 750 nM ABT-737 for 72 hours, followed by immunoblotting analysis for monitoring caspase-9 (Casp-9) cleavage (left lower panels), Bax translocation (to mitochondria, right upper panels), and cytochrome c (Cyt c) release (to cytosol, right lower panels). Bak was probed for loading control of the mitochondria-enriched fraction. The vertical lines indicate the splice site in the composite image derived from a single blot. (C) RPMI8226 cells were stably transfected with HA-tagged human full-length Bim (inset, using an anti-HA antibody [αHA]) or an empty vector (EV). Cells were then exposed to 500 to 750 nM ABT-737 for 48 hours and 72 hours, after which the percentage of apoptotic cells was determined by flow cytometry. Numbers indicate P values. (D) Bortezomib-resistant U266 cells (PS-R) were transiently transfected with an empty vector or HA-tagged Bim construct. After 48 hours, untreated cells were lysed and subjected to immunoblotting analysis for HA-Bim (inset, using αHA) or flow cytometry to determine the percentage of apoptotic (annexin V+) cells. IB, immunoblot; FITC, fluorescein isothiocyanate; L.E., long exposure; M.E., medium exposure; S.E., short exposure; UT, untreated.
Figure 3
Figure 3
Upregulation of Bim by SBHA potentiates ABT-737 lethality in bortezomib-resistant MM cells. (A-D) Bortezomib-resistant PS-R or 8226/VR cells were treated with ABT-737 (PS-R, 500 nM; 8226/VR, 300 nM) with or without the indicated concentrations (μM) of SBHA for 48 hours or 24 hours, respectively. After drug treatment, immunoblotting analysis and flow cytometry were performed to determine (A,B) levels of Bim and cleavage of caspases and PARP or (C,D) percentage of apoptosis. (E) Primary CD138+ cells were isolated from a bone marrow sample from a patient with relapsed MM. Cells were then treated with the indicated concentrations of ABT-737 (nM) with or without SBHA (μM) for 24 hours, after which flow cytometry was conducted to monitor percentage of early (annexin V+/PI–) and late (annexin V+/PI+) apoptosis. Values indicate the percentage of total annexin V+ cells. Parallel experiments were performed in additional primary samples derived from newly diagnosed MM patients or relapsed patients after treatment with bortezomib (arrowhead) (n = 6; 3 each for diagnosis and R patients). P < .05 for combination treatment vs each single agent. A+S, ABT-737 + SBHA; PI, propidium iodide.
Figure 4
Figure 4
Bim upregulated by SBHA attenuates ABT-737–induced autophagy. (A) Drug-naïve (U266) and bortezomib-resistant (PS-R) cells were treated with 500 nM ABT-737 with or without 20 μM SBHA for 16 hours, followed by immunoblotting analysis to monitor LC3 processing. (B) U266 and PS-R cells stably expressing GFP-LC3 were treated (6 hours) as described in (A), and then analyzed for GFP-LC3 puncta by confocal microscopy. Scale bar = 10 mm (magnification × 20). (C) U266 cells were treated as described in (A), after which cells were lysed in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer and subjected to immunoprecipitation (IP) with anti-Bcl-2 antibody and subsequent immunoblotting analyses using anti–Beclin-1 (Becl-1) and anti-Bim antibodies. (D) Primary CD138+ cells derived from a patient with relapsed MM were treated with 100 nM ABT-737 with or without 10 μM SBHA for 16 hours, after which immunofluorescence staining was performed to monitor expression of Bim or LC3 (both AlexaFluor 488, green) in CD138-phycoerythrin-positive cells. Scale bar = 10 μm; magnification ×40. (E) Tumor tissues obtained from PS-R cell mouse xenografts following the indicated treatments were subjected to immunoblotting analysis to monitor expression of Bim and LC3, as well as cleavage of caspase-9 and PARP. H, heavy chain; Ig, immunoglobulin; L, light chain.
Figure 5
Figure 5
Bim is required for disruption of autophagy and induction of apoptosis induced by ABT-737. (A) U266 cells stably transfected with Bim or scrambled sequence shRNA were incubated with 300 nM ABT-737 with or without 20 μM SBHA for 24 hours. Following treatment, immunoblotting analysis was performed to monitor Bim expression, LC3 processing, and PARP cleavage. (B) RPMI8226 cells stably transfected with LAMP2 (two subclones designated E3 and C3) or scrambled sequence shRNA (inset) were exposed to 300 nM ABT-737 with or without 20 μM SBHA for 24 hours followed by flow cytometry to determine the percentage of apoptotic cells. (C) MEFs derived from wild-type (Bim+/+) or bim knockout (Bim−/−) mice were treated with 500 nM ABT-737 with or without 20 μM SBHA for 16 hours, after which cells were stained with acridine orange (AO). (D) MEFs were exposed to 500 nM ABT-737 with or without 20 μM SBHA in the presence or absence of 50 μM CQ for 24 hours, after which cells were stained with annexin V-FITC. For both 5C and 5D images were captured by inverted fluorescence microscopy. BF, bright field. Scale bar = 10 μm; magnification ×10. S+A, SBHA + ABT-737; shL2, shLAMP2.
Figure 6
Figure 6
CQ disrupts autophagy and sensitizes Bimlow MM cells to the HDACI/BH3 mimetic regimen. (A) H929 cells were exposed to 300 nM ABT-737 with or without 30 μM SBHA in the presence or absence of 50 μM CQ, after which immunoblotting analysis was performed to monitor LC3 processing, Bim expression, and PARP cleavage. (B) In parallel, flow cytometry was performed to determine the percentage of apoptotic H929 cells. (C) Alternatively, cells were lysed in CHAPS buffer and subjected to co-IP (IP with anti-Bcl-2, immunoblotting with anti-Bim). IP without anti-Bcl-2 (lane 1), cell lysate (lane 2), or both (lane 3) was performed as controls. (D) Similarly, PS-R cells were incubated with relatively lower concentrations of ABT-737 (300 nM) with or without SBHA (15 μM) with or without 50 μM CQ for 48 hours, followed by flow cytometry to monitor apoptosis. (E) Primary CD138+ cells derived from newly diagnosed (displaying low basal Bim levels, inset) or relapsed MM patients (n = 4; 2 samples each for Dx and R) were treated with 100 nM ABT-737 plus 10 μM SBHA in the presence or absence of 10 μM CQ for 24 hours, after which flow cytometry was performed to determine the percentage of viable cells (7-aminoactinomycin D [7AAD] staining-negative).
Figure 7
Figure 7
The HDACI/BH3-mimetic regimen displays activity in vivo which is blocked by Bim shRNA knockdown. (A-B) NOD/SCID-γ mice were subcutaneously inoculated in the flank with (A) 5 × 106 PS-R or (B) U266/shBim cells. Doses of 100 mg/kg ABT-737 and 200 mg/kg SBHA were administrated intraperitoneally individually or in combination (n = 5 per group) 3 days per week. Control animals were administered equal volumes of vehicle. Tumor size was measured by caliper, and volumes were calculated by using the formula (length × width2)/2. (C) A mechanistic model for the roles of Bim in adaptive drug resistance and priming resistant MM cells toward death. Left: BH3 mimetics (eg, ABT-737) simultaneously activate apoptosis and autophagy by releasing Bim and Beclin-1 from Bcl-2, respectively. Whereas the former action is therapeutically beneficial, it is opposed by the latter cytoprotective response (autophagy ON), which raises the cell death threshold and promotes drug resistance. In this setting, Bim downregulation is associated with adaptive resistance to targeted agents such as bortezomib. Right: HDACIs upregulate Bim in MM cells, including those resistant to bortezomib due to Bim downregulation, and thereby reprime them to BH3-mimetic–induced apoptosis. The latter event is related, at least in part, to disruption of cytoprotective autophagy (autophagy OFF) through release of Bcl-2 from Bim and resulting enhanced sequestration of Beclin-1 by Bcl-2. Notably, inhibition of autophagy (eg, by CQ) significantly increases HDACI/BH3 mimetic regimen lethality, particularly in Bimlow MM cells. Thus, loss of Bim expression can contribute to an adaptive form of bortezomib resistance, and a Bim-targeting strategy combining HDACIs, which upregulate Bim, with BH3 mimetics, which release Bim from antiapoptotic Bcl-2 family proteins, may represent an effective approach to this problem.
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