Figure 5
Figure 5. EZN-3042 down-regulates survivin and synergizes with chemotherapy to overcome drug resistance in ALL in vitro. (A) Primary ALL xenografts of various cytogenetic types were cocultured with OP9 cells and assayed for in vitro sensitivity to survivin targeting EZN-3042 and scrambled control EZN-3088 over a period of 14 days. Viability was measured by Trypan blue exclusion of dead cells. Mean viability for EZN-3088–treated xenografts was 76.8% ± 15.9% (n = 5). EZN-3042–treated cells had a mean viability of 46.5% ± 10.5% (n = 5), representing a 30.3% significantly lower mean viability than controls (P < .00003). (B) Survivin protein knock-down of EZN-3042–treated ALL cells was confirmed by Western blotting. (C) Primary ALL (SFO2) was cocultured with OP9 cells and treated with nilotinib (200nM), a tyrosine kinase inhibitor (TKI), alone or in combination with EZN-3088 (Ctrl.) or EZN-3042, an antisense oligonucleotide (ASO). Drug and media were replaced on days 7 and 14, respectively. Viability was assessed by Trypan blue exclusion and is expressed as a percentage of the initial cell count. (D) Western blot analysis of survivin protein levels for cells shown in panel C. MNCs indicates normal PBMCs. (E) Representative annexin V–staining analysis of SFO2 ALL cells by FACS on days 9 and 15 after treatment in vitro (n = 3, supplemental Figure 6B). (F) SFO3 cells were cocultured with OP9 cells and treated with VDL (V, 1nM; D, 10pM; and L, 0.001 IU) alone or in combination with EZN-3088 (Ctrl.) or EZN-3042, an antisense oligonucleotide (ASO). Viability was determined on the days noted by Trypan blue exclusion. (G) Western blot of survivin protein levels of cells in panel F. (H) The CI for ED50, ED75, and ED90 was calculated using Chou and Talalay median effects analysis of primary ALL SFO3. The CI was determined to be < 1, indicating synergism with CI values for ED50 = 0.06 ± 0.05, ED75 = 0.061 ± 0.043, and ED90 = 0.058 ± 0.039. (I) LAX7R cells were cocultured with OP9 cells and treated with VDL (V, 1nM; D, 10pM; and L, 0.001 IU) alone, or in combination with EZN-3088 (Ctrl.) or EZN-3042, and antisense oligonucleotide (ASO). Viability was determined on the days noted by Trypan blue exclusion. (J) Western blot analysis of treated cells.

EZN-3042 down-regulates survivin and synergizes with chemotherapy to overcome drug resistance in ALL in vitro. (A) Primary ALL xenografts of various cytogenetic types were cocultured with OP9 cells and assayed for in vitro sensitivity to survivin targeting EZN-3042 and scrambled control EZN-3088 over a period of 14 days. Viability was measured by Trypan blue exclusion of dead cells. Mean viability for EZN-3088–treated xenografts was 76.8% ± 15.9% (n = 5). EZN-3042–treated cells had a mean viability of 46.5% ± 10.5% (n = 5), representing a 30.3% significantly lower mean viability than controls (P < .00003). (B) Survivin protein knock-down of EZN-3042–treated ALL cells was confirmed by Western blotting. (C) Primary ALL (SFO2) was cocultured with OP9 cells and treated with nilotinib (200nM), a tyrosine kinase inhibitor (TKI), alone or in combination with EZN-3088 (Ctrl.) or EZN-3042, an antisense oligonucleotide (ASO). Drug and media were replaced on days 7 and 14, respectively. Viability was assessed by Trypan blue exclusion and is expressed as a percentage of the initial cell count. (D) Western blot analysis of survivin protein levels for cells shown in panel C. MNCs indicates normal PBMCs. (E) Representative annexin V–staining analysis of SFO2 ALL cells by FACS on days 9 and 15 after treatment in vitro (n = 3, supplemental Figure 6B). (F) SFO3 cells were cocultured with OP9 cells and treated with VDL (V, 1nM; D, 10pM; and L, 0.001 IU) alone or in combination with EZN-3088 (Ctrl.) or EZN-3042, an antisense oligonucleotide (ASO). Viability was determined on the days noted by Trypan blue exclusion. (G) Western blot of survivin protein levels of cells in panel F. (H) The CI for ED50, ED75, and ED90 was calculated using Chou and Talalay median effects analysis of primary ALL SFO3. The CI was determined to be < 1, indicating synergism with CI values for ED50 = 0.06 ± 0.05, ED75 = 0.061 ± 0.043, and ED90 = 0.058 ± 0.039. (I) LAX7R cells were cocultured with OP9 cells and treated with VDL (V, 1nM; D, 10pM; and L, 0.001 IU) alone, or in combination with EZN-3088 (Ctrl.) or EZN-3042, and antisense oligonucleotide (ASO). Viability was determined on the days noted by Trypan blue exclusion. (J) Western blot analysis of treated cells.

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