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Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma.

Chen L, Rousseau RF, Middleton SA, Nichols GL, Newell DR, Lunec J, Tweddle DA - Oncotarget (2015)

Bottom Line: Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells.Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic.Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone.

View Article: PubMed Central - PubMed

Affiliation: Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle, United Kingdom.

ABSTRACT
Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

No MeSH data available.


Related in: MedlinePlus

(A) Sensitivity of a panel of neuroblastoma cell lines of varying MYCN, MDM2, p53 and p14ARF status to RG7388 treatment for 72 hours. p53 wt cell lines are significantly more sensitive to RG7388 treatment versus p53 mutant cell lines (Mann Whitney test, P < 0.0001), and Tet21N MYCN+ cells are significantly more sensitive to RG7388 compared with Tet21N MYCN− cells (paired t test, P < 0.005). Data are shown as the average of at least 3 independent experiments and error bars represent SEM. (B) The sensitivity of Tet21N MYCN+ and MYCN− cells to MDM2 antagonists, Nutlin-3a, NDD0005 and MI-63. Tet21N MYCN+ cells are significantly more sensitive to Nutlin-3a (paired t test, P < 0.05), NDD0005 (paired t test, P < 0.005) and MI-63 (paired t test, P < 0.05) treatment for 72 hours compared with Tet21N MYCN− cells. Data shown are the average of at least 3 independent experiments and error bars represent SEM. (C) RG7388 treatment leads to stabilisation of p53 and upregulation of p53 targets, MDM2, p21 and PUMA in p53 wt but not p53 mutant neuroblastoma cell lines. Western analysis for activation of the p53 pathway in the panel of neuroblastoma cell lines and the p53 wt MYCN regulatable SHEP Tet21N cells in response to treatment for 6 hours with 1× and 10× their respective RG7388 GI50 concentrations. p53 mutant cell lines are represented in bold font and MDM2 amplified cell lines are represented in italics. D, DMSO treated control cells.
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Figure 1: (A) Sensitivity of a panel of neuroblastoma cell lines of varying MYCN, MDM2, p53 and p14ARF status to RG7388 treatment for 72 hours. p53 wt cell lines are significantly more sensitive to RG7388 treatment versus p53 mutant cell lines (Mann Whitney test, P < 0.0001), and Tet21N MYCN+ cells are significantly more sensitive to RG7388 compared with Tet21N MYCN− cells (paired t test, P < 0.005). Data are shown as the average of at least 3 independent experiments and error bars represent SEM. (B) The sensitivity of Tet21N MYCN+ and MYCN− cells to MDM2 antagonists, Nutlin-3a, NDD0005 and MI-63. Tet21N MYCN+ cells are significantly more sensitive to Nutlin-3a (paired t test, P < 0.05), NDD0005 (paired t test, P < 0.005) and MI-63 (paired t test, P < 0.05) treatment for 72 hours compared with Tet21N MYCN− cells. Data shown are the average of at least 3 independent experiments and error bars represent SEM. (C) RG7388 treatment leads to stabilisation of p53 and upregulation of p53 targets, MDM2, p21 and PUMA in p53 wt but not p53 mutant neuroblastoma cell lines. Western analysis for activation of the p53 pathway in the panel of neuroblastoma cell lines and the p53 wt MYCN regulatable SHEP Tet21N cells in response to treatment for 6 hours with 1× and 10× their respective RG7388 GI50 concentrations. p53 mutant cell lines are represented in bold font and MDM2 amplified cell lines are represented in italics. D, DMSO treated control cells.

Mentions: The concentration of RG7388 required to inhibit growth by 50% (GI50) was determined using XTT cell proliferation assays in a panel of neuroblastoma cell lines, including 5 p53 mutant and 16 p53 wt cell lines of varying MYCN, MDM2 and p14ARF status, together with the p53 wt MYCN− regulatable SHEP Tet21N cells (Table 1, Figure 1A, Supplementary Figure 1A). The panel included 2 isogenic paired p53 wt and mutant cell lines, IMR32 and IMR/KAT100, and NGP, N_N20R1 and N_M5R1. p53 wt, MDM2 amplified human osteosarcoma SJSA-1 cells, previously shown to be sensitive to RG7388 and extensively used in the pre-clinical evaluation of several classes of MDM2-p53 antagonists to date, were used as a positive control [6, 8, 14-17] (Table 1). Consistent with the mechanism of action of MDM2-p53 antagonists, p53 wt neuroblastoma cell lines were significantly more sensitive to RG7388 compared to p53 mutant cell lines (P < 0.0001, Mann-Whitney test). Overall, all 16 neuroblastoma cell lines with wt p53 had nanomolar range GI50 values (range 14.8-140.3 nM; 68.2 (mean) ± 43.3 (SD) nM) of comparable sensitivity to SJSA-1 cells. In contrast, all 5 p53 mutant cell lines had GI50 values greater than 10 μM (range 10.1-16.9 μM; 14.6 (mean) ± 2.7 (SD) μM) (Table 1 and Figure 1A), representing > 200-fold differential between the average GI50 concentrations of p53 wt versus p53 mutant cell lines. Comparisons of GI50 concentrations between paired isogenic p53 wt and mutant neuroblastoma cell lines, demonstrated a 252-fold differential between IMR32 and IMR/KAT100, and a 406-fold and 384-fold differential between NGP and N_N20R1, and NGP and N_M5R1, respectively.


Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma.

Chen L, Rousseau RF, Middleton SA, Nichols GL, Newell DR, Lunec J, Tweddle DA - Oncotarget (2015)

(A) Sensitivity of a panel of neuroblastoma cell lines of varying MYCN, MDM2, p53 and p14ARF status to RG7388 treatment for 72 hours. p53 wt cell lines are significantly more sensitive to RG7388 treatment versus p53 mutant cell lines (Mann Whitney test, P < 0.0001), and Tet21N MYCN+ cells are significantly more sensitive to RG7388 compared with Tet21N MYCN− cells (paired t test, P < 0.005). Data are shown as the average of at least 3 independent experiments and error bars represent SEM. (B) The sensitivity of Tet21N MYCN+ and MYCN− cells to MDM2 antagonists, Nutlin-3a, NDD0005 and MI-63. Tet21N MYCN+ cells are significantly more sensitive to Nutlin-3a (paired t test, P < 0.05), NDD0005 (paired t test, P < 0.005) and MI-63 (paired t test, P < 0.05) treatment for 72 hours compared with Tet21N MYCN− cells. Data shown are the average of at least 3 independent experiments and error bars represent SEM. (C) RG7388 treatment leads to stabilisation of p53 and upregulation of p53 targets, MDM2, p21 and PUMA in p53 wt but not p53 mutant neuroblastoma cell lines. Western analysis for activation of the p53 pathway in the panel of neuroblastoma cell lines and the p53 wt MYCN regulatable SHEP Tet21N cells in response to treatment for 6 hours with 1× and 10× their respective RG7388 GI50 concentrations. p53 mutant cell lines are represented in bold font and MDM2 amplified cell lines are represented in italics. D, DMSO treated control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: (A) Sensitivity of a panel of neuroblastoma cell lines of varying MYCN, MDM2, p53 and p14ARF status to RG7388 treatment for 72 hours. p53 wt cell lines are significantly more sensitive to RG7388 treatment versus p53 mutant cell lines (Mann Whitney test, P < 0.0001), and Tet21N MYCN+ cells are significantly more sensitive to RG7388 compared with Tet21N MYCN− cells (paired t test, P < 0.005). Data are shown as the average of at least 3 independent experiments and error bars represent SEM. (B) The sensitivity of Tet21N MYCN+ and MYCN− cells to MDM2 antagonists, Nutlin-3a, NDD0005 and MI-63. Tet21N MYCN+ cells are significantly more sensitive to Nutlin-3a (paired t test, P < 0.05), NDD0005 (paired t test, P < 0.005) and MI-63 (paired t test, P < 0.05) treatment for 72 hours compared with Tet21N MYCN− cells. Data shown are the average of at least 3 independent experiments and error bars represent SEM. (C) RG7388 treatment leads to stabilisation of p53 and upregulation of p53 targets, MDM2, p21 and PUMA in p53 wt but not p53 mutant neuroblastoma cell lines. Western analysis for activation of the p53 pathway in the panel of neuroblastoma cell lines and the p53 wt MYCN regulatable SHEP Tet21N cells in response to treatment for 6 hours with 1× and 10× their respective RG7388 GI50 concentrations. p53 mutant cell lines are represented in bold font and MDM2 amplified cell lines are represented in italics. D, DMSO treated control cells.
Mentions: The concentration of RG7388 required to inhibit growth by 50% (GI50) was determined using XTT cell proliferation assays in a panel of neuroblastoma cell lines, including 5 p53 mutant and 16 p53 wt cell lines of varying MYCN, MDM2 and p14ARF status, together with the p53 wt MYCN− regulatable SHEP Tet21N cells (Table 1, Figure 1A, Supplementary Figure 1A). The panel included 2 isogenic paired p53 wt and mutant cell lines, IMR32 and IMR/KAT100, and NGP, N_N20R1 and N_M5R1. p53 wt, MDM2 amplified human osteosarcoma SJSA-1 cells, previously shown to be sensitive to RG7388 and extensively used in the pre-clinical evaluation of several classes of MDM2-p53 antagonists to date, were used as a positive control [6, 8, 14-17] (Table 1). Consistent with the mechanism of action of MDM2-p53 antagonists, p53 wt neuroblastoma cell lines were significantly more sensitive to RG7388 compared to p53 mutant cell lines (P < 0.0001, Mann-Whitney test). Overall, all 16 neuroblastoma cell lines with wt p53 had nanomolar range GI50 values (range 14.8-140.3 nM; 68.2 (mean) ± 43.3 (SD) nM) of comparable sensitivity to SJSA-1 cells. In contrast, all 5 p53 mutant cell lines had GI50 values greater than 10 μM (range 10.1-16.9 μM; 14.6 (mean) ± 2.7 (SD) μM) (Table 1 and Figure 1A), representing > 200-fold differential between the average GI50 concentrations of p53 wt versus p53 mutant cell lines. Comparisons of GI50 concentrations between paired isogenic p53 wt and mutant neuroblastoma cell lines, demonstrated a 252-fold differential between IMR32 and IMR/KAT100, and a 406-fold and 384-fold differential between NGP and N_N20R1, and NGP and N_M5R1, respectively.

Bottom Line: Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells.Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic.Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone.

View Article: PubMed Central - PubMed

Affiliation: Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle, United Kingdom.

ABSTRACT
Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

No MeSH data available.


Related in: MedlinePlus