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. 2008 Aug 1;112(3):793-804.
doi: 10.1182/blood-2007-10-116376. Epub 2008 May 27.

Vorinostat synergistically potentiates MK-0457 lethality in chronic myelogenous leukemia cells sensitive and resistant to imatinib mesylate

Yun Dai  1 , Shuang Chen, Charis A Venditti, Xin-Yan Pei, Tri K Nguyen, Paul Dent, Steven Grant
Affiliations

V体育官网 - Vorinostat synergistically potentiates MK-0457 lethality in chronic myelogenous leukemia cells sensitive and resistant to imatinib mesylate

V体育2025版 - Yun Dai et al. Blood. .

Abstract

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl(+) leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34(+) chronic myelogenous leukemia (CML) cells and Ba/F3 cells bearing various Bcr/Abl mutations (ie, T315I, E255K, and M351T), as well as IM-resistant K562 cells exhibiting Bcr/Abl-independent, Lyn-dependent resistance. These events were associated with inactivation and down-regulation of wild-type (wt) and mutated Bcr/Abl (particularly T315I) VSports手机版. Moreover, treatment with MK-0457 resulted in accumulation of cells with 4N or more DNA content, whereas coadministration of vorinostat markedly enhanced aurora kinase inhibition by MK-0457, and preferentially killed polyploid cells. Furthermore, vorinostat also interacted with a selective inhibitor of aurora kinase A and B to potentiate apoptosis without modifying Bcr/Abl activity. Finally, vorinostat markedly induced Bim expression, while blockade of Bim induction by siRNA dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. Together, these findings indicate that vorinostat strikingly increases MK-0457 activity against IM-sensitive and -resistant CML cells through inactivation of Bcr/Abl and aurora kinases, as well as by induction of Bim. .

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Figures

Figure 1
Figure 1
Vorinostat interacts synergistically with MK-0457 to induce apoptosis in CML cell lines. (A) K562 and LAMA84 cells were exposed to 1 μM MK-0457 (MK) for 24 hours (LAMA84) or 48 hours (K562), after which the percentage of cells exhibiting “low” Δψm or 7AAD positivity was determined by flow cytometry. (B) K562 and LAMA84 cells were treated as described in panel A, after which cells were lysed and subjected to Western blot using the indicated antibodies. (C) K562 and LAMA84 cells were incubated with increasing concentrations (5-300 nM) of MK in the absence or presence of vorinostat (Vor K562, 2 μm; LAM484, 1.5 μm) for 48 hours (K562) or 24 hours (LAMA84), after which the percentage of 7AAD+ cells was determined by flow cytometry. (D) K562 and LAMA84 cells were incubated with increasing concentrations (0.2-1 μM) of vorinostat in the absence or presence of 100 nM MK for 72 hours (K562) or 48 hours (LAMA84), after which the percentage of 7AAD+ cells was determined by flow cytometry. (E) K562 (top panel) and LAMA84 cells (bottom panel) were treated with a range of MK and Vor concentrations alone and in combination for 48 hours (K562) or 24 hours (LAMA84) at a fixed ratio as indicated. At the end of this period, the percentage of 7AAD+ cells was determined by flow cytometry; fractional effect values were determined by comparing results with those of untreated controls, and median dose effect analysis was used to characterize the nature of the interaction. Combination index (CI) values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (F) K562 and LAMA84 cells were treated with 5 to 100 nM MK with or without Vor (K562, 2 μM; LAMA84, 1.5 μM), after which Western blot analysis was performed to monitor cleavage of caspase-3 and PARP. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate (A,C,D). For each condition involving combined treatment, the net increase over treatment with Vor (C) or MK (D) alone was determined and evaluated for significance using the Student t test. Asterisks indicate significantly greater than values for treatment of cells with the single agent at the same concentrations (*P < .05; **P < .01). For panels B and F, each lane was loaded with 30 μg protein; blots were stripped and reprobed with β-actin or α-tubulin antibodies to ensure equal loading and transfer of protein. CF indicates cleavage fragment; Veh, vehicle. Two additional studies yielded equivalent results.
Figure 2
Figure 2
Vorinostat potentiates the lethality of MK-0457 in primary CML cells, while largely sparing normal bone marrow MNCs. (A) MNCs were isolated from bone marrow samples obtained from 2 patients with CML in chronic phase (patients 1 and 2) who had progressed after IM treatment, and from an additional patient (patient 3) who had not yet been treated, as well as from a normal bone marrow specimen. MNCs were exposed to 300 nM MK with or without 1.5 μM Vor for 48 hours, after which the percentage of 7AAD+ cells was determined by flow cytometry. (B) MNCs from patient 2 were treated with 100 to 500 nM MK with or without 1.5 μM Vor for 48 hours, after which cells were lysed and subjected to Western blot analysis to monitor caspase-3 cleavage. (C,D) Primary CD34+ CML cells isolated from bone marrow MNCs were obtained from patients 1 and 3. Cells were then stained with PE-conjugated CD34 antibody and subjected to flow cytometry to determine the purity of CD34+ cells. A representative result is shown (C). Numbers reflect the percentage of cells in the 2 top quadrants. Isolated primary CD34+ CML cells as well as normal CD34+ cells were exposed to 300 nM MK with or without 1.5 μM Vor for 24 to 48 hours, after which cells were stained with both DiOC6 and 7AAD and subjected to flow cytometry. “Low” DiOC6 uptake/7AAD− (bottom left quadrant) corresponds to early apoptosis (mitochondrial damage, reflected by loss of Δψm) and “low” DiOC6/7AAD+ (top left quadrant) corresponds to late apoptosis. Values reflect the percentage of cells in the corresponding quadrants. Results represent the means plus or minus SD for experiments performed in triplicate (A). (B) Each lane was loaded with 100 μg protein. The results of a representative experiment are shown; an additional experiment yielded equivalent results. Veh indicates vehicle.
Figure 3
Figure 3
The MK-0457/vorinostat regimen induces apoptosis in cells resistant to IM through disparate mechanisms. (A) IM-resistant K562-R cells were established as described in “Cells and reagents,” and Western blot analysis was performed to monitor expression of Bcr/Abl and phospho-CrkL, as well as levels of total and phosphorylated Lyn (inset). Parental K562 and K562-R cells were exposed to 5 μM MK for 48 hours, after which the percentage of 7AAD+ cells was determined by flow cytometry. (B) K562-R cells were incubated with 0.1 to 1 μM MK with or without 1.5 μM Vor for 48 hours, after which flow cytometry was performed to monitor the percentage of 7AAD+ cells. (C) K562R cells were treated as described in panel B, after which Western blot analysis was performed to monitor expression of total and phosphorylated Lyn, as well as processing of caspase-3 and cleavage of PARP. (D) Ba/F3 cells bearing wt or various point mutations, including T351I, M351T, and E255K, were exposed to 100 to 500 nM MK-0457 with or without 1.5 μM vorinostat for 48 hours, after which the percentage of 7AAD+ cells was determined by flow cytometry. (E) Alternatively, cells were lysed and subjected to Western blot analysis to evaluate cleavage of caspase-9 and PARP. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate (A,B,D). For each condition involving combined treatment, the net increase compared with treatment with Vor alone was determined and the significance of differences evaluated using the Student t test. Asterisks indicate significantly greater than values obtained after treatment of cells with MK alone at the same concentrations (*P < .05; **P < .01). For panels A inset, C, and E, each lane was loaded with 30 μg protein; blots were stripped and reprobed with antibodies to β-actin or α-tubulin to ensure equal loading and transfer. CF indicates cleavage fragment; Veh, vehicle. Two additional studies yielded equivalent results.
Figure 4
Figure 4
Coadministration of MK-0457 and vorinostat results in inactivation and down-regulation of wt and T315I Bcr/Abl kinases. (A) K562 and LAMA84 cells were incubated with 5 to 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours (K562) or 24 hours (LAMA84). (B) Ba/F3 cells with wt or T315I Bcr/Abl were treated with 0.1 to 0.5 μM MK with or without 1.5 μM Vor for 48 hours. (A,B) After drug treatment, cells were lysed, and Western blot analysis was performed to assess expression of total and phosphorylated forms of Bcr/Abl and CrkL. Each lane was loaded with 30 μg protein; blots were stripped and reprobed with antibodies to β-actin to ensure equal loading and transfer. Results are representative of 3 separate experiments. Protein bands of phospho-CrkL were quantified using an imaging system as described in “Western blot.” Values reflect the ratio of integrated densitometric determinations between untreated and drug-treated cells. Veh indicates vehicle. Two additional studies yielded equivalent results.
Figure 5
Figure 5
Vorinostat enhances inhibition of aurora kinases by MK-0457 in IM-sensitive and -resistant cells, including those bearing the T315I mutation. (A) K562 and LAMA84 cells were exposed to 5 to 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours (K562) or 24 hours (LAMA84). (B) IM-resistant K562-R cells were treated with 0.1 to 1 μM MK with or without 1.5 μM Vor for 48 hours. (C) Ba/F3 cells with wt or T315I Bcr/Abl were treated with 0.1 to 0.5 μM MK with or without 1.5 μM Vor for 48 hours. (A-C) After drug treatment, Western blot analysis was performed to monitor expression of total and phosphorylated (Ser 10) histone H3, as well as total aurora A and B. Each lane was loaded with 30 μg protein; blots were stripped and reprobed with antibodies to β-actin to ensure equal loading and transfer. Two additional studies yielded equivalent results.
Figure 6
Figure 6
Aurora kinase inhibition contributes functionally to the lethality of MK-0457/vorinostat in CML cells. (A) K562 (top) and LAMA84 (bottom) cells were treated with 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 24 hours (K562) or 16 hours (LAMA84), after which cytospin slides were prepared and stained with FITC-conjugated antibodies to phosphorylated (Ser 10) histone H3 (top panels) as described in “Immunocytochemistry.” Bottom panels exhibit DAPI staining. Images were captured with an Olympus BX40 microscope at 20×/0.50 (Olympus America, Center Valley, PA) and a CE digital camera (Alpha Innotech, San Leandro, CA) with RS Image software version 1.7.3. (Roper Scientific Photometrics, Tucson, AZ). (B) LAMA84 and K562 cells were incubated with 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 16 hours (LAMA84) or 24 and 48 hours (K562), after which cells were stained with PI and DNA content was analyzed by flow cytometry. N indicates number of ploidy. (C) K562 cells were exposed to 10 μM of a selective aurora kinase A and B inhibitor (Aur K inh) in the absence or the presence of Vor (2 μM) for 48 hours, after which cells were lysed and subjected to Western blot to monitor expression of phospho-Bcr/Abl and phospho-CrkL, as well as levels of total and phosphorylated histone H3. Each lane was loaded with 30 μg protein. (D) K562 and LAMA84 cells were treated with 10 μM Aur K inh with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours, after which the percentage of 7AAD+ cells were determined by flow cytometry. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate. Asterisks indicate significantly greater than the value for treatment of cells with Aur K inh alone at same concentrations (**P < .01). The results of a representative experiment are shown (A-C); 2 additional studies yielded equivalent results.
Figure 7
Figure 7
Induction of Bim by vorinostat plays a functional role in interactions between vorinostat and MK-0457. (A) K562 (top) and LAMA84 (bottom) cells were treated with 5 to 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours (K562) or 24 hours (LAMA84). (B) IM-resistant K562-R cells were treated with 0.1 to 1 μM MK with or without 1.5 μM Vor for 48 hours. (C) Ba/F3 cells with wt or T315I Bcr/Abl were treated with 0.1 to 0.5 μM MK with or without 1.5 μM Vor for 48 hours. (D) MNCs from bone marrow samples obtained from CML patients 1 and 3 were exposed to 100 to 500 nM MK with or without 1.5 μM Vor for 48 hours. (A-D) After drug treatment, cells were lysed and subjected to Western blot analysis to monitor expression of Bim. (E) K562 cells were stably transfected with constructs encoding siRNA against Bim (siBim) or nonspecific sequence (siCtrl). Cells were exposed to 5 μM of Vor (top panel) or MK (bottom panel) for 48 hours, after which cells were stained with annexin V–FITC and subjected to flow cytometry. (F) K562 cells transfected with siBim or siCtrl were treated with 100 nM MK with or without 2 μM Vor for 48 hours, after which cells were lysed and Western blot analysis was performed to monitor expression of Bim, Bcr/Abl, and phosphohistone H3. (G) Alternatively, cells were stained with DiOC6 (top panels) or annexin V–FITC (bottom panels), respectively, and subjected to flow cytometry. Each lane was loaded with 30 μg (A-C,F) or 100 μg (D) of protein; blots were stripped and reprobed with β-actin or α-tubulin antibodies to ensure equal loading and transfer of protein. Two additional studies yielded equivalent results. The results of a representative experiment are shown (E,G); 2 additional studies yielded equivalent results.
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