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TP53 loss creates therapeutic vulnerability in colorectal cancer.

Liu Y, Zhang X, Han C, Wan G, Huang X, Ivan C, Jiang D, Rodriguez-Aguayo C, Lopez-Berestein G, Rao PH, Maru DM, Pahl A, He X, Sood AK, Ellis LM, Anderl J, Lu X - Nature (2015)

Bottom Line: Previous clinical applications of α-amanitin have been limited owing to its liver toxicity.However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity.Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

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The sensitivity of POLR2Aloss cells to POLR2A inhibition is independent of p53a, Protein levels of POLR2A, p53 and EpCAM in a panel of isogenic human xhCRC cell lines. b, Cell proliferation of isogenic xhCRC cells treated with α-Amanitin. c, Sensitivity of isogenic xhCRC cells to 5-FU, Oxaliplatin (Oxa) or SN-38 treatment combined with or without α-Amanitin. d, Cell proliferation of isogenic xhCRC cells treated with Ama-HEA125. Data are mean and s.d. of three independent experiments in the figure.
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Figure 3: The sensitivity of POLR2Aloss cells to POLR2A inhibition is independent of p53a, Protein levels of POLR2A, p53 and EpCAM in a panel of isogenic human xhCRC cell lines. b, Cell proliferation of isogenic xhCRC cells treated with α-Amanitin. c, Sensitivity of isogenic xhCRC cells to 5-FU, Oxaliplatin (Oxa) or SN-38 treatment combined with or without α-Amanitin. d, Cell proliferation of isogenic xhCRC cells treated with Ama-HEA125. Data are mean and s.d. of three independent experiments in the figure.

Mentions: Blockage of RNA polymerase may lead to p53 accumulation and activation19. To examine the effects of p53, we recapitulated the concomitant deletion of TP53 and POLR2A in HCT116 and xhCRC cell lines (Fig. 3a and Extended Data Fig. 6a–c). The xhCRC cell line (TP53+/+;POLR2A+/+), established from a freshly isolated xenografted human colon tumour, demonstrated enhanced tumourigenicity in vivo20. Except for slightly increased cell proliferation, no significant changes in their sensitivity to α-Amanitin were observed in either xhCRC or HCT116 cells with hemizygous deletion of TP53. By contrast, hemizygous loss of POLR2A markedly sensitized these cells to α-Amanitin treatment regardless of their TP53 status (Fig. 3b and Extended Data Fig. 6d–g). The mRNA synthesis activity of the RNA polymerase II is essential to any type of cells including therapy-resistant tumour cells. We examined the drug sensitivity of POLR2Aneutral and POLR2Aloss cells to three major chemotherapy drugs for CRC: 5-fluorouracil (5-FU), oxaliplatin, and SN-38. Inhibition of POLR2A by α-Amanitin significantly enhanced cell-killing effects of all three drugs in the POLR2Aloss xhCRC cells, but had no notable effects on the POLR2Aneutral cells (Fig. 3c), suggesting therapeutic vulnerability of POLR2Aloss colorectal tumours. Free α-Amanitin is toxic to liver due to its interaction with OATP1B3, a transporter exclusively expressed on the membrane of hepatocytes10. However, α-Amanitin, when conjugated with specific antibodies, is no longer a substrate for OATP1B310,11,21. This strategy overcomes the toxicity of α-Amanitin for clinical applications. We used α-Amanitin conjugated to a monoclonal antibody (HEA125) against EpCAM, a cancer antigen overexpressed in adenocarcinomas11,22. The Ama-HEA125 conjugate selectively killed the POLR2Aloss xhCRC and HCT116 cancer cells in a p53-independent manner and reduced the effective doses of α-Amanitin by at least 10,000-fold (IC50 ~ 0.01 ng ml−1) in vitro (Fig. 3d and Extended Data Fig. 6h).


TP53 loss creates therapeutic vulnerability in colorectal cancer.

Liu Y, Zhang X, Han C, Wan G, Huang X, Ivan C, Jiang D, Rodriguez-Aguayo C, Lopez-Berestein G, Rao PH, Maru DM, Pahl A, He X, Sood AK, Ellis LM, Anderl J, Lu X - Nature (2015)

The sensitivity of POLR2Aloss cells to POLR2A inhibition is independent of p53a, Protein levels of POLR2A, p53 and EpCAM in a panel of isogenic human xhCRC cell lines. b, Cell proliferation of isogenic xhCRC cells treated with α-Amanitin. c, Sensitivity of isogenic xhCRC cells to 5-FU, Oxaliplatin (Oxa) or SN-38 treatment combined with or without α-Amanitin. d, Cell proliferation of isogenic xhCRC cells treated with Ama-HEA125. Data are mean and s.d. of three independent experiments in the figure.
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Figure 3: The sensitivity of POLR2Aloss cells to POLR2A inhibition is independent of p53a, Protein levels of POLR2A, p53 and EpCAM in a panel of isogenic human xhCRC cell lines. b, Cell proliferation of isogenic xhCRC cells treated with α-Amanitin. c, Sensitivity of isogenic xhCRC cells to 5-FU, Oxaliplatin (Oxa) or SN-38 treatment combined with or without α-Amanitin. d, Cell proliferation of isogenic xhCRC cells treated with Ama-HEA125. Data are mean and s.d. of three independent experiments in the figure.
Mentions: Blockage of RNA polymerase may lead to p53 accumulation and activation19. To examine the effects of p53, we recapitulated the concomitant deletion of TP53 and POLR2A in HCT116 and xhCRC cell lines (Fig. 3a and Extended Data Fig. 6a–c). The xhCRC cell line (TP53+/+;POLR2A+/+), established from a freshly isolated xenografted human colon tumour, demonstrated enhanced tumourigenicity in vivo20. Except for slightly increased cell proliferation, no significant changes in their sensitivity to α-Amanitin were observed in either xhCRC or HCT116 cells with hemizygous deletion of TP53. By contrast, hemizygous loss of POLR2A markedly sensitized these cells to α-Amanitin treatment regardless of their TP53 status (Fig. 3b and Extended Data Fig. 6d–g). The mRNA synthesis activity of the RNA polymerase II is essential to any type of cells including therapy-resistant tumour cells. We examined the drug sensitivity of POLR2Aneutral and POLR2Aloss cells to three major chemotherapy drugs for CRC: 5-fluorouracil (5-FU), oxaliplatin, and SN-38. Inhibition of POLR2A by α-Amanitin significantly enhanced cell-killing effects of all three drugs in the POLR2Aloss xhCRC cells, but had no notable effects on the POLR2Aneutral cells (Fig. 3c), suggesting therapeutic vulnerability of POLR2Aloss colorectal tumours. Free α-Amanitin is toxic to liver due to its interaction with OATP1B3, a transporter exclusively expressed on the membrane of hepatocytes10. However, α-Amanitin, when conjugated with specific antibodies, is no longer a substrate for OATP1B310,11,21. This strategy overcomes the toxicity of α-Amanitin for clinical applications. We used α-Amanitin conjugated to a monoclonal antibody (HEA125) against EpCAM, a cancer antigen overexpressed in adenocarcinomas11,22. The Ama-HEA125 conjugate selectively killed the POLR2Aloss xhCRC and HCT116 cancer cells in a p53-independent manner and reduced the effective doses of α-Amanitin by at least 10,000-fold (IC50 ~ 0.01 ng ml−1) in vitro (Fig. 3d and Extended Data Fig. 6h).

Bottom Line: Previous clinical applications of α-amanitin have been limited owing to its liver toxicity.However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity.Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Show MeSH
Related in: MedlinePlus