Figure 1.
Antileukemic effect of the combination of ABT-869 with SAHA on leukemia cell lines with FLT3-ITD mutations.
Combination indices (CIs) quantitatively describe the interactions between ABT-869 and SAHA in MV4-11 cells (A), MOLM-14 cells (B). The X-axis shows inhibitory effect by the combination of two drugs and Y-axis shows the CI values. ED stands for effective dosage. The CI values at ED50, ED75 and ED90 values of two drugs were inserted into the figures. These results were generated by CalcuSyn software. (C) Percentage of apoptosis induced by ABT-869 alone, SAHA alone, and combination treatment for 48 hours. The experiments were in triplicate. Comb 1*: Combination of ABT-869 3 nM and SAHA 2 µM, p<0.001 as compared to either single drug treatment. Comb 2**: ABT-869 6 nM+SAHA 4 µM, p<0.001 as compared to either single drug treatment. (D) Western blot analysis of cleaved PARP in MV4-11 and MOLM-14 cells. β-actin was used as loading control. Cells were treated with either control, ABT-869 3 nM for MV4-11 and 6 nM for MOLM-14, SAHA 4 µM or combination therapy for 48 hours, and then followed by lysis and Western blot analysis.
Figure 2.
Combination treatment enhances loss of clonogenic survival and apoptosis of AML cells.
(A) MV4-11 cells were treated with DMSO control, ABT-869 3 nM, SAHA 2 µM for 48 hours. (B) MOLM-14 cells were treated with DMSO control, ABT-869 6 nM, SAHA 2 µM for 48 hours. In both (A) and (B), cells were washed and plated in HSC-CFU media for 7 days. Representative pictures were presented in the right panel. The colony number was expressed as mean ± SD of average of 5 random 40 times fields. φp<0.01 as compared to single drug treatment. (C) Primary AML cells with FLT3-ITD mutation (left panel) or wt-FLT3 (right panel) were incubated with DMSO, ABT-869 20 nM, SAHA 4 µM and combination for 48 hours, followed by FACS analysis of apoptosis. The bar graphs show percentage of apoptotic cells. The experiments were duplicated. *p<0.001 as compared to single drug treatment in all the three primary AML patient samples with FLT3-ITD mutation.
Table 1.
List of core gene signature identified by Affymetrix microarray studies of MV4-11 and MOLM-14 cells treated with combination of ABT-869 and SAHA.
Figure 3.
Impact of modulation of PRL-3 expression on drug sensitivity.
(A) MOLM-14 cells were treated with DMSO, ABT-869 6 nM, SAHA 4 µM or combination therapy for 48 hours, and then followed by lyses and Western blot analysis of PRL-3, p-Stat5, Stat5. Immunoprecipitation (IP) was performed using anti-p-Tyrosine antibody, followed by Western blot with anti-FLT3 antibody. The same blot was then stripped and reprobed with anti-FLT3 antibody. The upper band is160 kDa glycosylated mature FLT3 and the lower band is 130 kDa nonglycosylated FLT3. (B) MOLM-14 cells were transfected with vector control (Mock) or pLVX-puro-PRL3 vector. Cells were treated with either ABT-869 6 nM, SAHA 4 µM or combination therapy for 48 hours. (C) MOLM-14 cells were transfected with control and PRL-3 shRNA-0780 respectively for 48 hours, and then treated with ABT-869 3 nM, SAHA 2 µM or combination therapy for 72 hours. Apoptosis assay was used to determine the cell viability in different treatments. (D) Flow cytomertric analysis of apoptosis in MOLM-14 cells were treated with either DMSO control or PRL-3 inhibitor I at concentration 2 and 10 µM for 48 h. (E) Effect of PRL-3 inhibitor I on PRL-3 protein expression. Apoptosis data shown represents the means of three independent experiments ± SD (*p<0.01, **p<0.05).
Figure 4.
Comparisons of a panel of anti-apoptotic proteins and signaling transduction pathways in MOLM-14-Mock and MOLM-14-PRL3 cells.
The two isogenic cells were lysed and subjected to 10% to 12% SDS-PAGE. Western blots were detected with the indicated antibodies for the assessment of expression level changes in anti-apoptotic proteins (A) and PI3K/Akt, MAPK/ERK and Stat pathways (B). β-actin was used as a loading control. Arrow highlighted Mcl-1.
Figure 5.
The association of PRL-3 and FLT3-ITD in AML.
(A) Western blot assessment of PRL-3 protein in two isogenic cell lines, TF-1 and TF1-ITD. β-actin was used as loading control. (B) TF1-ITD cells were treated with DMSO, ABT-869 2 nM and 5 nM for 48 hours, and then followed by IP and Western blot analysis as described in Fig. 4.A. (C) MOLM-14 cells were treated with DMSO control or IDR E804 100 nM for 48 h. The cells were then lysed, and analysed by immunoblotting using anti-p-Stat5, anti-Stat5, anti-PRL-3 clone 318 and anti-β-actin. (D) MOLM-14 cell lysate was immunoprecipitated with anti-PRL-3 or anti-IgG antibody. The immunoprecipitates were immunoblotted with anti-HDAC4 antibody. (E) DLD-1-GFP-hPRL3 cell lysate was immunoprecipitated with anti-GFP or ant-IgG antibody, followed by immunoblotting analysis with anti-HDAC4 antibody. Total cellular extracts (inputs) were used as positive controls. (F) Western blot analysis of cell lysates extracted from MOLM-14 cells treated with DMSO, SAHA 4 µM, MG-132 10 µM or SAHA plus MG-132. The incubation duration of DMSO and SAHA was 24 h and MG-132 was added 4 h before harvest of cells. Densitometric analysis was performed using Amersham Image Scanner with LabScan ImageQuant TL Software. The level of PRL-3 was normalized with each β-actin level.
Figure 6.
Evaluation of PRL-3 protein expression in bone marrow sections from primary AML patients and normal donors by IHC.
(A) A representative image from nine PRL-3 positive AML cases was shown. PRL-3 positive signals were detected in the cytoplasm of myeloid cells. (B) A representative result from ten PRL-3 negative AML cases was shown. (C) A normal bone marrow image from 6 donors was presented. The original magnifications were 63×.
Figure 7.
Schematic representation of the molecular mechanisms of enhanced downregulation of PRL-3 by combination treatment with ABT-869 and SAHA.
ABT-868-induced inhibition of PRL-3 is due to targeting upstream FLT3-ITD and Stat pathway. HDAC4 physically interacts with PRL-3. SAHA mediated degradation of PRL-3 is dependent on the proteasome pathway.