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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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Suppression of POLR2A selectively inhibits the POLR2Aloss tumour growtha, b, Gross tumour images (a) and growth curves (b) of xenograft tumours derived from subcutaneously implanted HCT116 or SNU283 cells expressing control or Dox-inducible POLR2A shRNA. n = 5 mice per group. Error bars, s.e.m. c–e, Tumour growth curves (c, **p < 0.01, error bars, s.e.m.), gross tumour images (d) and weights (e, error bars, s.d.) of xenograft tumours derived from orthotopically implanted POLR2Aneutral and POLR2Aloss HCT116 cells expressing Dox-inducible control or POLR2A shRNA. n = 5 mice per group. f–i, Representative bioluminescent images (f, h)and tumour growth curves (g, i)of orthotopic xenograft tumours derived from POLR2Aneutral and POLR2Aloss HCT116 (f, g) or xhCRC cells (h, i) that received dual intraperitoneal injections of HEA125 antibody or Ama-HEA125 antibody-drug conjugate (3, 10, 30 and 90 μg kg−1). n = 10 mice per group. Error bars, s.e.m.
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Figure 4: Suppression of POLR2A selectively inhibits the POLR2Aloss tumour growtha, b, Gross tumour images (a) and growth curves (b) of xenograft tumours derived from subcutaneously implanted HCT116 or SNU283 cells expressing control or Dox-inducible POLR2A shRNA. n = 5 mice per group. Error bars, s.e.m. c–e, Tumour growth curves (c, **p < 0.01, error bars, s.e.m.), gross tumour images (d) and weights (e, error bars, s.d.) of xenograft tumours derived from orthotopically implanted POLR2Aneutral and POLR2Aloss HCT116 cells expressing Dox-inducible control or POLR2A shRNA. n = 5 mice per group. f–i, Representative bioluminescent images (f, h)and tumour growth curves (g, i)of orthotopic xenograft tumours derived from POLR2Aneutral and POLR2Aloss HCT116 (f, g) or xhCRC cells (h, i) that received dual intraperitoneal injections of HEA125 antibody or Ama-HEA125 antibody-drug conjugate (3, 10, 30 and 90 μg kg−1). n = 10 mice per group. Error bars, s.e.m.

Mentions: To test the anti-tumour effect of POLR2A inhibition in vivo, HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA were injected subcutaneously into NOD/SCID mice. Following initial tumour establishment, administration of Dox (1.0 μg ml−1) in drinking water suppressed POLR2A expression and consequently inhibited the growth of SNU283-derived tumours (Fig. 4a, b and Extended Data Fig. 7a). However, no substantial differences were observed between control and POLR2A-knockdown HCT116-derived tumours. Histopathologic analyses demonstrated that POLR2A-knockdown SNU283 tumours had significantly reduced cell proliferation, but more apoptotic cells, as compared with the corresponding control tumours (Extended Data Fig. 7b, c). By contrast, no significant changes were observed in the control or POLR2A-knockdown HCT116 tumours. However, hemizygous deletion of POLR2A sensitized HCT116-derived tumours to Dox treatment (Extended Data Figure 7d, e). Next, we employed an orthotopic tumour model by injecting POLR2Aneutral and POLR2Aloss HCT116 cells into the cecal wall of NOD/SCID mice. In-vivo tumour imaging demonstrated that Dox-induced POLR2A inhibition led to a significant decrease in tumour growth kinetics in the POLR2Aloss tumours, but not in the control POLR2Aneutral tumours (Fig. 4c–e, and Extended Data Fig. 7f). To further test the efficacy of POLR2A silencing in vivo, we used a nanoliposomal delivery platform, DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), for systemic delivery of POLR2A siRNAs23 (Extended Data Fig. 8a, b). Following four weeks of systemic therapy, compared with control siRNA-DOPC treatment, mice in the 125 μg kg−1 of POLR2A siRNA-DOPC treatment groups had pronounced growth reduction of POLR2Aloss tumors, while POLR2Aneutral tumors only had significant growth inhibition even at the dose of 1,000 μg kg−1 (Extended Data Fig. 8c–h).


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)

Suppression of POLR2A selectively inhibits the POLR2Aloss tumour growtha, b, Gross tumour images (a) and growth curves (b) of xenograft tumours derived from subcutaneously implanted HCT116 or SNU283 cells expressing control or Dox-inducible POLR2A shRNA. n = 5 mice per group. Error bars, s.e.m. c–e, Tumour growth curves (c, **p < 0.01, error bars, s.e.m.), gross tumour images (d) and weights (e, error bars, s.d.) of xenograft tumours derived from orthotopically implanted POLR2Aneutral and POLR2Aloss HCT116 cells expressing Dox-inducible control or POLR2A shRNA. n = 5 mice per group. f–i, Representative bioluminescent images (f, h)and tumour growth curves (g, i)of orthotopic xenograft tumours derived from POLR2Aneutral and POLR2Aloss HCT116 (f, g) or xhCRC cells (h, i) that received dual intraperitoneal injections of HEA125 antibody or Ama-HEA125 antibody-drug conjugate (3, 10, 30 and 90 μg kg−1). n = 10 mice per group. Error bars, s.e.m.
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Figure 4: Suppression of POLR2A selectively inhibits the POLR2Aloss tumour growtha, b, Gross tumour images (a) and growth curves (b) of xenograft tumours derived from subcutaneously implanted HCT116 or SNU283 cells expressing control or Dox-inducible POLR2A shRNA. n = 5 mice per group. Error bars, s.e.m. c–e, Tumour growth curves (c, **p < 0.01, error bars, s.e.m.), gross tumour images (d) and weights (e, error bars, s.d.) of xenograft tumours derived from orthotopically implanted POLR2Aneutral and POLR2Aloss HCT116 cells expressing Dox-inducible control or POLR2A shRNA. n = 5 mice per group. f–i, Representative bioluminescent images (f, h)and tumour growth curves (g, i)of orthotopic xenograft tumours derived from POLR2Aneutral and POLR2Aloss HCT116 (f, g) or xhCRC cells (h, i) that received dual intraperitoneal injections of HEA125 antibody or Ama-HEA125 antibody-drug conjugate (3, 10, 30 and 90 μg kg−1). n = 10 mice per group. Error bars, s.e.m.
Mentions: To test the anti-tumour effect of POLR2A inhibition in vivo, HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA were injected subcutaneously into NOD/SCID mice. Following initial tumour establishment, administration of Dox (1.0 μg ml−1) in drinking water suppressed POLR2A expression and consequently inhibited the growth of SNU283-derived tumours (Fig. 4a, b and Extended Data Fig. 7a). However, no substantial differences were observed between control and POLR2A-knockdown HCT116-derived tumours. Histopathologic analyses demonstrated that POLR2A-knockdown SNU283 tumours had significantly reduced cell proliferation, but more apoptotic cells, as compared with the corresponding control tumours (Extended Data Fig. 7b, c). By contrast, no significant changes were observed in the control or POLR2A-knockdown HCT116 tumours. However, hemizygous deletion of POLR2A sensitized HCT116-derived tumours to Dox treatment (Extended Data Figure 7d, e). Next, we employed an orthotopic tumour model by injecting POLR2Aneutral and POLR2Aloss HCT116 cells into the cecal wall of NOD/SCID mice. In-vivo tumour imaging demonstrated that Dox-induced POLR2A inhibition led to a significant decrease in tumour growth kinetics in the POLR2Aloss tumours, but not in the control POLR2Aneutral tumours (Fig. 4c–e, and Extended Data Fig. 7f). To further test the efficacy of POLR2A silencing in vivo, we used a nanoliposomal delivery platform, DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), for systemic delivery of POLR2A siRNAs23 (Extended Data Fig. 8a, b). Following four weeks of systemic therapy, compared with control siRNA-DOPC treatment, mice in the 125 μg kg−1 of POLR2A siRNA-DOPC treatment groups had pronounced growth reduction of POLR2Aloss tumors, while POLR2Aneutral tumors only had significant growth inhibition even at the dose of 1,000 μg kg−1 (Extended Data Fig. 8c–h).

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