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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

RG7388 treatment induces cell cycle arrest and/or apoptosis in p53 wt neuroblastoma cell linesSub-G1 and cell cycle phase distribution (A) and G1:S ratios (B) of 8 p53 wt neuroblastoma cell lines and the MYCN regulatable SHEP Tet21N cells treated for 24 hours with 1×, 10×, 50× or 100× their respective RG7388 GI50 concentrations. (C) Caspase 3/7 activity in the same panel of cell lines in response to 24 hours treatment with 1× or 10× their respective RG7388 GI50 concentrations or an equal volume of DMSO. Data are expressed as fold change relative to DMSO control, and are shown as the average of at least 3 independent experiments and error bars represent SEM. D, DMSO treated control cells; MYCN+, Tet21N MYCN+; MYCN−, Tet21N MYCN−.
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Figure 2: RG7388 treatment induces cell cycle arrest and/or apoptosis in p53 wt neuroblastoma cell linesSub-G1 and cell cycle phase distribution (A) and G1:S ratios (B) of 8 p53 wt neuroblastoma cell lines and the MYCN regulatable SHEP Tet21N cells treated for 24 hours with 1×, 10×, 50× or 100× their respective RG7388 GI50 concentrations. (C) Caspase 3/7 activity in the same panel of cell lines in response to 24 hours treatment with 1× or 10× their respective RG7388 GI50 concentrations or an equal volume of DMSO. Data are expressed as fold change relative to DMSO control, and are shown as the average of at least 3 independent experiments and error bars represent SEM. D, DMSO treated control cells; MYCN+, Tet21N MYCN+; MYCN−, Tet21N MYCN−.

Mentions: From the original panel of cell lines assessed above, 8 p53 wt neuroblastoma cell lines (non-MYCN amplified SHSY5Y & SKNRA; MYCN amplified IMR32 & LAN5; MDM2 and MYCN co-amplified NGP & NB1691; p14ARF methylated Per-108 & GIMEN) and Tet21N cells were analysed for cell cycle phase distribution and induction of apoptosis in response to RG7388 (Figure 2 and Supplementary Table 1). Cells were treated for 24 hours with 1×, 10×, 50× and 100× their respective GI50 concentrations of RG7388 (Table 1) and analysed using flow cytometry. An increase in the percentage of sub-G1 events, as a surrogate marker of apoptosis, was observed in all cell lines in response to one or more concentrations of RG7388. Overall, the accumulation of events in sub-G1 phase occurred in a concentration-dependent manner (Figure 2A and Supplementary Table 1). The G1:S ratio was calculated as an indicator of G1 cell cycle arrest, and with the exception of MYCN amplified LAN5 and IMR32 cells, all other cell lines including Tet21N cells in the presence and absence of MYCN, demonstrated at least a 2-fold increase in G1:S ratio in response to treatment with at least one or more concentrations of RG7388 (Figure 2B and Supplementary Table 1). In line with the role of MYCN in driving proliferation, switching off MYCN in the Tet21N system led to an increase in baseline G1:S ratio (MYCN+, 4.2 ± 0.8 versus MYCN−, 17.6 ± 5.3) (Figure 2B and Supplementary Table 1). Finally, treatment with ≥ 10× GI50 concentrations of RG7388 led to an accumulation of cells in G2/M phase in NGP, NB1691 and GIMEN cells (Figure 2A and Supplementary Table 1).


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)

RG7388 treatment induces cell cycle arrest and/or apoptosis in p53 wt neuroblastoma cell linesSub-G1 and cell cycle phase distribution (A) and G1:S ratios (B) of 8 p53 wt neuroblastoma cell lines and the MYCN regulatable SHEP Tet21N cells treated for 24 hours with 1×, 10×, 50× or 100× their respective RG7388 GI50 concentrations. (C) Caspase 3/7 activity in the same panel of cell lines in response to 24 hours treatment with 1× or 10× their respective RG7388 GI50 concentrations or an equal volume of DMSO. Data are expressed as fold change relative to DMSO control, and are shown as the average of at least 3 independent experiments and error bars represent SEM. D, DMSO treated control cells; MYCN+, Tet21N MYCN+; MYCN−, Tet21N MYCN−.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: RG7388 treatment induces cell cycle arrest and/or apoptosis in p53 wt neuroblastoma cell linesSub-G1 and cell cycle phase distribution (A) and G1:S ratios (B) of 8 p53 wt neuroblastoma cell lines and the MYCN regulatable SHEP Tet21N cells treated for 24 hours with 1×, 10×, 50× or 100× their respective RG7388 GI50 concentrations. (C) Caspase 3/7 activity in the same panel of cell lines in response to 24 hours treatment with 1× or 10× their respective RG7388 GI50 concentrations or an equal volume of DMSO. Data are expressed as fold change relative to DMSO control, and are shown as the average of at least 3 independent experiments and error bars represent SEM. D, DMSO treated control cells; MYCN+, Tet21N MYCN+; MYCN−, Tet21N MYCN−.
Mentions: From the original panel of cell lines assessed above, 8 p53 wt neuroblastoma cell lines (non-MYCN amplified SHSY5Y & SKNRA; MYCN amplified IMR32 & LAN5; MDM2 and MYCN co-amplified NGP & NB1691; p14ARF methylated Per-108 & GIMEN) and Tet21N cells were analysed for cell cycle phase distribution and induction of apoptosis in response to RG7388 (Figure 2 and Supplementary Table 1). Cells were treated for 24 hours with 1×, 10×, 50× and 100× their respective GI50 concentrations of RG7388 (Table 1) and analysed using flow cytometry. An increase in the percentage of sub-G1 events, as a surrogate marker of apoptosis, was observed in all cell lines in response to one or more concentrations of RG7388. Overall, the accumulation of events in sub-G1 phase occurred in a concentration-dependent manner (Figure 2A and Supplementary Table 1). The G1:S ratio was calculated as an indicator of G1 cell cycle arrest, and with the exception of MYCN amplified LAN5 and IMR32 cells, all other cell lines including Tet21N cells in the presence and absence of MYCN, demonstrated at least a 2-fold increase in G1:S ratio in response to treatment with at least one or more concentrations of RG7388 (Figure 2B and Supplementary Table 1). In line with the role of MYCN in driving proliferation, switching off MYCN in the Tet21N system led to an increase in baseline G1:S ratio (MYCN+, 4.2 ± 0.8 versus MYCN−, 17.6 ± 5.3) (Figure 2B and Supplementary Table 1). Finally, treatment with ≥ 10× GI50 concentrations of RG7388 led to an accumulation of cells in G2/M phase in NGP, NB1691 and GIMEN cells (Figure 2A and Supplementary Table 1).

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