Survivin promotes drug resistance. (A) 15 unsorted primary pre–B-ALL (1-15) and 3 unsorted primary T-ALL cases (16-18) and LPS-stimulated proliferating and unstimulated CD19⁺ B cells as controls (n = 3, supplemental Figure 1) were studied for survivin mRNA levels by qRT-PCR (top panel) and survivin protein expression by Western analysis (bottom panel). Major karyotypes or the primary cases are indicated at the top of the figure and are listed in supplemental Table 1. Survivin is significantly more expressed in primary ALL cells (n = 18) compared with normal CD19⁺ B-cell controls (n = 3; 3.46 ± 2.38 vs 0.26 ± 0.06, respectively; Survivin /GAPDH-fold increase; P < .02). *P < .05; no symbol indicates P < .005. (B) A human survivin promoter–driven GFP lentiviral reporter (survivin reporter-GFP) was used in LAX7R cells. Unsorted bulk ALL or ALL cells FACS sorted for GFP^High and GFP^Low were analyzed for survivin expression by qRT-PCR. *P < .05. (C) Unsorted, LAX7R cells transduced with the survivin reporter GFP were treated with either media control or a combination of VDL (V, 1nM; D, 0.1nM; and L, 0.01 IU) for 48 hours and analyzed for apoptosis by flow cytometry after annexin V costaining. (D) Viable annexin V⁻/7-AAD⁻ LAX7R cells after VDL or control treatment were further analyzed by flow cytometry for survivin reporter-GFP expression. (E) A lentiviral survivin IRES GFP (survivin-GFP) was used to overexpress survivin in ALL. Survivin-GFP ALL cells and empty-GFP controls were treated in vitro with V alone (left panel) or with the VDL combined therapy, as in panel D (right). Viability was assessed by MTT assay. (F) Primary ALL cells (LAX7R) cells transduced with either nonsilencing or survivin shRNA were treated with V at the indicated doses and viability was assessed with an MTT assay. Asterisks (*) denote statistical significance between survivin shRNA knock-down and controls.

Loss of survivin decreases self-renewal, sensitizes leukemia cells to chemotherapy, and abrogates leukemia. (A) A lentiviral survivin IRES GFP (survivin-GFP) was used to overexpress survivin in ALL. GFP fluorescence imaging of colonies from control and transduced ALL cells (100× magnification; left panel) and counts of colonies of primary ALL (LAX7R) and survivin-GFP–transduced cells after primary and secondary replating (middle and right panel). (B) Kaplan-Meier survival curves of NSG mice engrafted with survivin-GFP or empty-GFP transduced primary ALL cells. (C) Primary ALL cells (LAX7R) cells transduced with either nonsilencing or survivin shRNA. Colony images (10×; left panel) and colony counts of primary and secondary plating (middle right panel). (D) Survival of NSG mice recipients of chemotherapy (VDL) and primary ALL transduced with either nonsilencing or survivin shRNA. The treatment (Tx) period was 28 days. (E) Representative histologic sections from mice showing BM, spleen, liver, and lung tissue stained for human CD45 (brown; 400× magnification). (F) FACS analysis of splenocytes (SPC) and BM cells from nonsilencing + VDL- and survivin shRNA + VDL–treated mice, stained for human and murine CD45. (G) Human GAPDH and murine HPRT were detected by PCR in splenocytes and BM genomic DNA.

Conditional knockout of survivin abrogates leukemia in vitro. (A) Schematic of oncogenic transformation of IL-7–primed BM harvested from survivin floxed (survivin^fl/fl) mice transformed with BCR-ABL p210 retrovirus. Subsequent to leukemic outgrowth after cytokine withdrawal, cultured cells were transduced with either GFP control or the Cre-GFP vector to inducibly delete survivin. (B) Conditional knockout of survivin. The presence of floxed and deleted survivin alleles in transduced cells was confirmed by PCR of genomic DNA. (C) Cre-GFP- and GFP control–transduced BCR-ABL p210 murine leukemia cultured cells were monitored for GFP expression by flow cytometry. Statistical analyses were performed at 120 hours. P < .0006.

Sensitivity of primary ALL cells to survivin-targeting EZN-3042. (A-G) Dose sensitivity of primary ALL cells to single-agent survivin antisense EZN-3042 and scrambled control EZN-3088 was determined. ALL cells were cocultured with OP9 cells and treated for 7 days with single-agent doses ranging from 5-20μM of respective LNA. Cell viability was determined by Trypan blue exclusion of dead 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.

Combined EZN-3042 and chemotherapy treatment prolongs survival of recipient mice with primary ALL. (A) Bioimaging of primary ALL on day 56 after transplantation of the EZN-3042 + VDL–treated cohort (n = 10) and the EZN-3088 + VDL–treated control group (n = 11). (B) Kaplan-Meier curves of NSG mice engrafted with LAX7R cells and treated with saline (black long dotted line), EZN-3088 (black small dotted line), or EZN-3042 (red interrupted line) as single agents or either EZN-3088 (black line) or EZN-3042 (red line) administered IP or by subcutaneous pump, respectively, in combination with VDL (V, 0.5 mg/kg/d; D, 10.5 mg/kg/d; and L, 1500 IU/kg/d) for 28 days. MST was 38 days for the saline group (n = 3), 41 days for the EZN-3088 group (n = 3), and 39 days for the EZN-3042 group (n = 3). The 69-day MST of the EZN-3088 + VDL–treated group (n = 11; IP, n = 3; subcutaneous pump, n = 8) compares to the 75-day MST of EZN-3042 + VDL–treated group (n = 10; IP, n = 3; subcutaneous pump, n = 7). P < .04.