Limits...
Identifying the determinants of response to MDM2 inhibition.

Saiki AY, Caenepeel S, Cosgrove E, Su C, Boedigheimer M, Oliner JD - Oncotarget (2015)

Bottom Line: To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies.To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification.Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

View Article: PubMed Central - PubMed

Affiliation: Oncology Research, Amgen, Inc., Thousand Oaks, California, USA.

ABSTRACT
Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2-amplified subset of p53WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

No MeSH data available.


Related in: MedlinePlus

Four insensitive cell lines with wildtype TP53 genomic sequence are actually mutant(A) CAPAN-2, MDA-MB-453, MG-63, NCI-H82, and HCT116 cells were treated with either DMSO or 10 μM AMG 232 for 24 hours. Total protein lysates were collected, and immunoblot analysis of MDM2, p53, and p21 expression was performed. With the exception of HCT116, expression levels of these proteins were unaffected by MDM2 inhibition, suggesting that p53 was non-functional in these lines. Additionally, MDA-MB-453 lysates contained a faster migrating band, indicating truncation of p53 in that cell line. (B) Transcript sequencing of TP53 cDNA generated from CAPAN-2 and NCI-H82 showed aberrantly spliced mRNA due to a silent G > T transversion in the last nucleotide of exon 4 (*, Thr125). In CAPAN-2, the resulting transcript included the first 271 bp of intron 4 spliced to the final 240 bp of exon 11, with deletion of all exons in between. In NCI-H82, the aberrant transcript deleted the last 200 bp of exon 4 (aa 59–125).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480710&req=5

Figure 3: Four insensitive cell lines with wildtype TP53 genomic sequence are actually mutant(A) CAPAN-2, MDA-MB-453, MG-63, NCI-H82, and HCT116 cells were treated with either DMSO or 10 μM AMG 232 for 24 hours. Total protein lysates were collected, and immunoblot analysis of MDM2, p53, and p21 expression was performed. With the exception of HCT116, expression levels of these proteins were unaffected by MDM2 inhibition, suggesting that p53 was non-functional in these lines. Additionally, MDA-MB-453 lysates contained a faster migrating band, indicating truncation of p53 in that cell line. (B) Transcript sequencing of TP53 cDNA generated from CAPAN-2 and NCI-H82 showed aberrantly spliced mRNA due to a silent G > T transversion in the last nucleotide of exon 4 (*, Thr125). In CAPAN-2, the resulting transcript included the first 271 bp of intron 4 spliced to the final 240 bp of exon 11, with deletion of all exons in between. In NCI-H82, the aberrant transcript deleted the last 200 bp of exon 4 (aa 59–125).

Mentions: As part of the cell panel curation, we searched for drug sensitivity correlates that might reveal previously unrecognized confounders to the stratification analysis. Strikingly, we observed that the four least responsive p53WT cell lines (CAPAN-2, MDA-MB-453, MG-63, and NCI-H82) also displayed the lowest expression levels of TP53 transcript (Figure 2). Indeed, these 4 cell lines occupied a spatially distinct cluster in the plots of sensitivity vs. TP53 expression. To further investigate p53 expression in these cell lines, immunoblot analysis was performed following 24 hours of treatment with MDM2 inhibitor AMG 232 [6]. HCT116, a p53WT cell line that is sensitive to MDM2 inhibition, was used as a control in these experiments. As expected, AMG 232 treatment of HCT116 cells resulted in upregulation of MDM2 and p21 expression, as well as accumulation of p53 (Figure 3A). No such upregulation was seen in the other 4 cell lines, suggesting that p53 was non-functional in these lines. Additionally, in MDA-MB-453 cells, a band which migrated faster than the control was detected, indicative of a truncated mutant p53 protein, consistent with previously reported data (Figure 3A; [16]).


Identifying the determinants of response to MDM2 inhibition.

Saiki AY, Caenepeel S, Cosgrove E, Su C, Boedigheimer M, Oliner JD - Oncotarget (2015)

Four insensitive cell lines with wildtype TP53 genomic sequence are actually mutant(A) CAPAN-2, MDA-MB-453, MG-63, NCI-H82, and HCT116 cells were treated with either DMSO or 10 μM AMG 232 for 24 hours. Total protein lysates were collected, and immunoblot analysis of MDM2, p53, and p21 expression was performed. With the exception of HCT116, expression levels of these proteins were unaffected by MDM2 inhibition, suggesting that p53 was non-functional in these lines. Additionally, MDA-MB-453 lysates contained a faster migrating band, indicating truncation of p53 in that cell line. (B) Transcript sequencing of TP53 cDNA generated from CAPAN-2 and NCI-H82 showed aberrantly spliced mRNA due to a silent G > T transversion in the last nucleotide of exon 4 (*, Thr125). In CAPAN-2, the resulting transcript included the first 271 bp of intron 4 spliced to the final 240 bp of exon 11, with deletion of all exons in between. In NCI-H82, the aberrant transcript deleted the last 200 bp of exon 4 (aa 59–125).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480710&req=5

Figure 3: Four insensitive cell lines with wildtype TP53 genomic sequence are actually mutant(A) CAPAN-2, MDA-MB-453, MG-63, NCI-H82, and HCT116 cells were treated with either DMSO or 10 μM AMG 232 for 24 hours. Total protein lysates were collected, and immunoblot analysis of MDM2, p53, and p21 expression was performed. With the exception of HCT116, expression levels of these proteins were unaffected by MDM2 inhibition, suggesting that p53 was non-functional in these lines. Additionally, MDA-MB-453 lysates contained a faster migrating band, indicating truncation of p53 in that cell line. (B) Transcript sequencing of TP53 cDNA generated from CAPAN-2 and NCI-H82 showed aberrantly spliced mRNA due to a silent G > T transversion in the last nucleotide of exon 4 (*, Thr125). In CAPAN-2, the resulting transcript included the first 271 bp of intron 4 spliced to the final 240 bp of exon 11, with deletion of all exons in between. In NCI-H82, the aberrant transcript deleted the last 200 bp of exon 4 (aa 59–125).
Mentions: As part of the cell panel curation, we searched for drug sensitivity correlates that might reveal previously unrecognized confounders to the stratification analysis. Strikingly, we observed that the four least responsive p53WT cell lines (CAPAN-2, MDA-MB-453, MG-63, and NCI-H82) also displayed the lowest expression levels of TP53 transcript (Figure 2). Indeed, these 4 cell lines occupied a spatially distinct cluster in the plots of sensitivity vs. TP53 expression. To further investigate p53 expression in these cell lines, immunoblot analysis was performed following 24 hours of treatment with MDM2 inhibitor AMG 232 [6]. HCT116, a p53WT cell line that is sensitive to MDM2 inhibition, was used as a control in these experiments. As expected, AMG 232 treatment of HCT116 cells resulted in upregulation of MDM2 and p21 expression, as well as accumulation of p53 (Figure 3A). No such upregulation was seen in the other 4 cell lines, suggesting that p53 was non-functional in these lines. Additionally, in MDA-MB-453 cells, a band which migrated faster than the control was detected, indicative of a truncated mutant p53 protein, consistent with previously reported data (Figure 3A; [16]).

Bottom Line: To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies.To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification.Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

View Article: PubMed Central - PubMed

Affiliation: Oncology Research, Amgen, Inc., Thousand Oaks, California, USA.

ABSTRACT
Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2-amplified subset of p53WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

No MeSH data available.


Related in: MedlinePlus