Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia

Abstract

SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL.

Figure 1

Intrinsic activity of BV6 on BCP-ALL cell lines. (a) Heterogeneous sensitivity of BCP-ALL cell lines for cell death after exposure to BV6 (48 h). UoCB6 and REH display half-maximal inhibitory concentration (IC₅₀) values at nanomolar concentrations in contrast to Nalm-6 and RS4;11 showing sensitivity in the micromolar range. (b–e) Inhibition of BV6-induced cell death (48-h exposure to BV6 at indicated concentrations) by 20 μM zVAD.fmk (zVAD), 30 μM Necrostatin-1 (Nec-1), or 40 μg/ml etanercept (Et). Percentages of dead cells were estimated by flow cytometry according to forward and side scatter criteria, three independent experiments each performed in triplicate, estimation of IC₅₀ (a), comparison of BV6 to BV6 and the respective inhibitors; mean and S.D. are indicated; significance by Mann-Whitney U-test; *P<0.01 and ^#P<0.05 (b–e)

Figure 2

TNF-α signaling upon BV6 exposure. Western blot analysis of (a) cIAP1 degradation, (b) NIK accumulation and (c) NF-κB activation in BCP-ALL cell lines exposed to BV6 for indicated time points (UoCB6 and REH: 1 μM BV6; Nalm-6 and RS4;11: 10 μM BV6; or equivalent doses of DMSO for 24 h). For each analysis, one representative blot of three is shown. Arrows indicate NIK. (d) Increased TNF-α protein levels in cellular supernatants of BCP-ALL cell lines treated with BV6 or control as indicated (mean and S.D. of three independent experiments each performed in duplicate). (e–h) TNFR complex II formation upon BV6 exposure (UoCB6 and REH: 1 μM; Nalm-6 and RS4;11: 1 μM (+) or 10 μM (++) BV6) and dependency on TNF-α (with or without 40 μg/ml etanercept). Caspase-8 immunoprecipitation in the presence of 10 μM zVAD.fmk (UoCB6, REH and Nalm-6), and western blot analysis of indicated proteins (one representative experiment of three is shown)

Figure 3

Apoptosis signaling upon BV6 treatment. (a) Loss of mitochondrial membrane potential (MMP) in BCP-ALL cell lines upon BV6 treatment (UoCB6 and REH: 1μM; Nalm-6 and RS4;11: 10 μM BV6; 24 h, with or without 40 μg/ml etanercept; mean and S.D. of three independent experiments each performed in triplicate, significance by Mann-Whitney U-test, *P<0.01. (b) Caspase-3 activation in BCP-ALL cell lines upon BV6 treatment (UoCB6 and REH: 1 μM; Nalm-6 and RS4;11: 1 μM (+) or 10 μM (++) BV6; 24 h), and (c) TNF-α dependency of the BV6-sensitive leukemia cell lines UoCB6 and REH (additional treatment with or without 40 μg/ml etanercept), western blot analysis. (d) PARP cleavage upon BV6 treatment for indicated time points (UoCB6 and REH: 1 μM; Nalm-6 and RS4;11: 10 μM BV6), western blot analysis. For each western blot analysis, one representative experiment out of three is displayed

Figure 4

Dependency of BV6-induced cell death on RIP1. Block of BV6-induced cell death (exposure at indicated concentrations for 48 h) by RIP1 knockdown in BV6-sensitive (a and b) but not BV6-insensitive (c and d) cell lines. Mean and S.D. of three independent experiments each performed in triplicate are shown, significance by Mann-Whitney U-test; *P<0.01 and ^#P<0.05. (e) Blocked BV6-induced cell death (48-h exposure at indicated concentrations), and (f) loss of mitochondrial membrane potential (MMP; 24 h, 1 μM BV6) in REH cells with stable shRNA-mediated RIP1 knockdown. Mean and S.D. of three independent experiments each performed in triplicate are shown, significance by Mann-Whitney U-test; *P<0.01. (g) Efficient knockdown of RIP1 in shRNA versus control-transduced REH cells is shown by western blot analysis. (h) Absence of caspase-8 and -3 activation (1 μM BV6 for indicated time points) in REH cells upon shRNA-mediated RIP1 knockdown, as well as (i) impaired complex II formation (1 μM BV6 for 18 h, caspase-8 immunoprecipitation in the presence of 10 μM zVAD.fmk and western blot analysis of indicated proteins)

Figure 5

BV6-induced cell death in primary ALL. Cell death induction in primary, patient-derived BCP-ALL samples (n=40) by BV6 (A) and dependency on TNF-α (n=20 primografts; B). Cell death by flow cytometry according to forward and side scatter criteria upon treatment with 1 μM BV6 for 48 h and with 40 μg/ml (BV6+Et) or without, BV6-induced cell death was calculated as the difference of total and spontaneous cell death; experiments performed in triplicate; mean and S.D. are indicated. (C) Activation of caspase-3 (a), and cytochrome c release (b) upon BV6 treatment (1 μM, 18 h) in primograft samples. Percentages of cells with activated caspase-3 and cells with released cytochrome c were estimated by flow cytometry in triplicate, bars represent mean values and S.D.

Figure 6

Preclinical activity of BV6 on pediatric high-risk ALL. NOD/SCID mice transplanted with a high-risk BCP-ALL showing presence of human ALL cells in the peripheral blood were treated as indicated. (a) Leukemia load (numbers of human leukemia cells in the recipient's peripheral blood at the end of treatment, recipients individual and median values are shown). (b) Percentages of human (CD19 positive) ALL cells in the recipient's peripheral blood over time (data points represent mean values and S.D. per group). Superior leukemia-free survival of ALL-bearing animals after treatment with BV6 (c), a combination of VDA induction-chemotherapy and BV6 compared to chemotherapy alone (d), and in vivo TNF-α dependency of intrinsic (e) but not of chemo-sensitizing BV6 activity (f). Probabilities of leukemia-free survival after treatment with vehicle (V), BV6, induction-chemotherapy (vincristine, dexamethasone and asparaginase; VDA) or combinations thereof (10 animals per group) with or without etanercept (Et; five animals per group); TTR, time to leukemia reoccurrence; Kaplan-Meier analysis, log-rank test; P, significance