Abstract
The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirustype-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/ AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.
| Original language | English |
|---|---|
| Pages (from-to) | 22730-22736 |
| Number of pages | 7 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 116 |
| Issue number | 45 |
| DOIs | |
| Publication status | Published - 2019 |
Keywords
- Cervical cancer
- Copanlisib
- HER2/neu
- Neratinib
- PIK3CA
ASJC Scopus subject areas
- General
Access to Document
Other files and links
Fingerprint
Dive into the research topics of 'Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy'. Together they form a unique fingerprint.Cite this
- APA
- Standard
- Harvard
- Vancouver
- Author
- BIBTEX
- RIS
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 45, 2019, p. 22730-22736.
Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy
AU - Zammataro, Luca
AU - Lopez, Salvatore
AU - Bellone, Stefania
AU - Pettinella, Francesca
AU - Bonazzoli, Elena
AU - Perrone, Emanuele
AU - Zhao, Siming
AU - Menderes, Gulden
AU - Altwerger, Gary
AU - Han, Chanhee
AU - Zeybek, Burak
AU - Bianchi, Anna
AU - Manzano, Aranzazu
AU - Manara, Paola
AU - Cocco, Emiliano
AU - Buza, Natalia
AU - Hui, Pei
AU - Wong, Serena
AU - Ravaggi, Antonella
AU - Bignotti, Eliana
AU - Romani, Chiara
AU - Todeschini, Paola
AU - Zanotti, Laura
AU - Odicino, Franco
AU - Pecorelli, Sergio
AU - Donzelli, Carla
AU - Ardighieri, Laura
AU - Angioli, Roberto
AU - Raspagliesi, Francesco
AU - Scambia, Giovanni
AU - Choi, Jungmin
AU - Dong, Weilai
AU - Bilguvar, Kaya
AU - Alexandrov, Ludmil B.
AU - Silasi, Dan Arin
AU - Huang, Gloria S.
AU - Ratner, Elena
AU - Azodi, Masoud
AU - Schwartz, Peter E.
AU - Pirazzoli, Valentina
AU - Stiegler, Amy L.
AU - Boggon, Titus J.
AU - Lifton, Richard P.
AU - Schlessinger, Joseph
AU - Santin, Alessandro D.
N1 - Funding Information: 7. E. D. Pleasance et al., A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463, 191–196 (2010). Materials and Methods Patients and Specimens. Briefly, DNA and RNA fractions from consented patients were isolated from biological samples (i.e., fresh tumor specimens, primary cell lines, venous blood, primary fibroblasts, or frozen myometrium) using an AllPrep DNA/RNA Mini Kit (Qiagen) following the manufacturer’s procedure and prepared as described (6) (see SI Appendix, Materials and Methods for details). The study protocol was approved by the Yale Human Investigation Committee and was conducted in accordance with the Declaration of Helsinki. DNA was extracted from 54 primary tumors and 15 primary cell lines with limited passages. WES. Genomic DNA was captured and analyzed as described (4, 36–38). Identified variants were annotated based on novelty, impact on the encoded protein, conservation, and expression using an automated pipeline. See SI Appendix, Materials and Methods for details. Somatic Copy Number Variation. Copy number variants were identified by means of Excavator (version 2.1) (39) and GISTIC (version 2.0.16) (40) to calculate significantly amplified or deleted regions as previously described (39, 40). See SI Appendix, Materials and Methods for details. Real-Time Reverse Transcription-PCR (qRT-PCR). RNA isolation and quantita- tive RT-PCR were performed using standard protocols on the AB 7500 Real-Time PCR instrument. Primer sequences are described in SI Appendix. Drug Preparation and In Vitro Chemosensitivity Assay. Cells were exposed to afatinib (Boehringer) or neratinib (Puma) or copanlisib (Bayer), dissolved per the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO) as single agents or in combination, at concentrations ranging from 0.1 to 10 mM. After 72 h of incubation, cells were harvested for flow cytometric count. Detailed information is provided in SI Appendix, Materials and Methods. In Vivo Experiments in Tumors Harboring ERBB2/PI3K/AKT/mTOR Pathway Alterations. Briefly, HER2/neu-mutated (CVX-4) and PIK3CA-mutated (CVX-5) cell lines were injected into the s.c. region of 5-to 6-wk-old SCID mice (Envigo Dublin VA, Horst, Netherlands). The mice were divided into multiple treatment groups (i.e., controls, afatinib, neratinib, copanlisib, single agents, or combinations) and received vehicle (0.5% methylcellulose-0.4% Tween 80) or the drugs, as described in Figs. 3 and 4, and in SI Appendix, Materials and Methods. All of the mice were housed and treated in accordance with the policies set forth by the Yale Institutional Animal Care and Use Committee (IACUC). Statistical Analysis. All statistical analyses were performed using Prism 6 software (GraphPad Prism Software Inc., San Diego, CA). A P value of <0.05 was considered as the level of statistical significance. See SI Appendix, Materials and Methods for details. Data Availability. All data discussed in the paper are available in Dataset S1. ACKNOWLEDGMENTS. This work was supported in part by Gilead Sciences Inc. (Foster City, CA), Puma Biotechnology, Inc. (Los Angeles, CA), NIH Grants R01 CA154460-01 and U01 CA176067-01A1, the Deborah Bunn Alley Foundation, the Tina Brozman Foundation, the Discovery to Cure Foundation, and the Guido Berlucchi Foundation (to A.D.S.). This investigation was also supported by NIH Research Grant CA-16359 from the National Cancer Institute and by Stand-up-to Cancer (SU2C) Convergence Grant 2.0 (to A.D.S.). We thank Katerina Politi (Department of Pathology, Yale University) for generous support with reagents. Preliminary results for this work were presented at the 50th Annual Meeting of the Society of Gynecologic Oncology, March 16–19, 2019, Honolulu, HI (Poster ID 1215: “Whole exome sequencing (WES) reveals novel therapeutic targets in cervical cancer”, by Lopez S. et al.). 8. S. S. Krishna, I. Majumdar, N. V. Grishin, Structural classification of zinc fingers: Survey and summary. Nucleic Acids Res. 31, 532–550 (2003). 9. A. Gonzalez-Perez et al., IntOGen-mutations identifies cancer drivers across tumor types. Nat. Methods 10, 1081–1082 (2013). 10. D. R. Alessi, K. Sakamoto, J. R. Bayascas, LKB1-dependent signaling pathways. Annu. Rev. Biochem. 75, 137–163 (2006). 11. S. N. Wingo et al., Somatic LKB1 mutations promote cervical cancer progression. PLoS One 4, e5137 (2009). 12. D. Chen et al., ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 121, 1071–1083 (2005). 13. R. K. Pandya, J. R. Partridge, K. R. Love, T. U. Schwartz, H. L. Ploegh, A structural el-ement within the HUWE1 HECT domain modulates self-ubiquitination and substrate ubiquitination activities. J. Biol. Chem. 285, 5664–5673 (2010). 14. J. J. Yi et al., An autism-linked mutation disables phosphorylation control of UBE3A. Cell 162, 795–807 (2015). MEDICAL SCIENCES Downloaded at Elsevier Science London on November 6, 2019 15. L. Huang et al., Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321–1326 (1999). 16. H. B. Kamadurai et al., Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex. Mol. Cell 36, 1095–1102 (2009). 17. H. B. Kamadurai et al., Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. Elife 2, e00828 (2013). 18. J. N. Kloth et al., Expression of Smad2 and Smad4 in cervical cancer: Absent nuclear Smad4 expression correlates with poor survival. Mod. Pathol. 21, 866–875 (2008). 19. J. Garnon et al., Fragile X-related protein FXR1P regulates proinflammatory cytokine tumor necrosis factor expression at the post-transcriptional level. J. Biol. Chem. 280, 5750–5763 (2005). 20. T. K. Khera, A. D. Dick, L. B. Nicholson, Fragile X-related protein FXR1 controls post-transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-alpha production by transforming growth factor-beta1. FEBS J. 277, 2754–2765 (2010). 21. B. Hao, S. Oehlmann, M. E. Sowa, J. W. Harper, N. P. Pavletich, Structure of a Fbw7-Skp1-cyclin E complex: Multisite-phosphorylated substrate recognition by SCF ubiq-uitin ligases. Mol. Cell 26, 131–143 (2007). 22. M. Welcker, B. E. Clurman, FBW7 ubiquitin ligase: A tumour suppressor at the crossroads of cell division, growth and differentiation. Nat. Rev. Cancer 8, 83–93 (2008). 23. E. Cocco, S. Lopez, A. D. Santin, M. Scaltriti, Prevalence and role of HER2 mutations in cancer. Pharmacol. Ther. 199, 188–196 (2019). 24. R. Bose et al., Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov. 3, 224–237 (2013). 25. H. Greulich et al., Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2. Proc. Natl. Acad. Sci. U.S.A. 109, 14476–14481 (2012). 26. A. I. Ojesina et al., Landscape of genomic alterations in cervical carcinomas. Nature 506, 371–375 (2014). 27. Cancer Genome Atlas Research Network et al., Integrated genomic and molecular characterization of cervical cancer. Nature 543, 378–384 (2017). 28. L. B. Alexandrov et al., Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013). 29. S. Inoue et al., Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by pre-venting c-Myc/Miz1-mediated down-regulation of p21 and p15. Genes Dev. 27, 1101– 1114 (2013). 30. P. U. Freda et al., Analysis of GNAS mutations in 60 growth hormone secreting pi-tuitary tumors: Correlation with clinical and pathological characteristics and surgical outcome based on highly sensitive GH and IGF-I criteria for remission. Pituitary 10, 275–282 (2007). 31. J. Wu et al., Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development. Sci. Transl. Med. 3, 92ra66 (2011). 32. A. Matsubara et al., Lobular endocervical glandular hyperplasia is a neoplastic entity with frequent activating GNAS mutations. Am. J. Surg. Pathol. 38, 370–376 (2014). 33. S. P. Malkoski, X. J. Wang, Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer. FEBS Lett. 586, 1984–1992 (2012). 34. J. S. Frenel et al., Safety and efficacy of pembrolizumab in advanced, programmed death ligand 1-positive cervical cancer: Results from the phase Ib KEYNOTE-028 trial. J. Clin. Oncol. 35, 4035–4041 (2017). 35. A. B. Hanker, V. Kaklamani, C. L. Arteaga, Challenges for the clinical development of PI3K inhibitors: Strategies to improve their impact in solid tumors. Cancer Discov. 9, 482–491 (2019). 36. K. Cibulskis et al., Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013). 37. H. Li, R. Durbin, Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009). 38. H. Li et al.; 1000 Genome Project Data Processing Subgroup, The sequence alignment/ map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). 39. A. Magi et al., EXCAVATOR: Detecting copy number variants from whole-exome se-quencing data. Genome Biol. 14, R120 (2013). 40. R. Beroukhim et al., Assessing the significance of chromosomal aberrations in cancer: Methodology and application to glioma. Proc. Natl. Acad. Sci. U.S.A. 104, 20007– 20012 (2007). Funding Information: This work was supported in part by Gilead Sciences Inc. (Foster City, CA), Puma Biotechnology, Inc. (Los Angeles, CA), NIH Grants R01 CA154460-01 and U01 CA176067-01A1, the Deborah Bunn Alley Foundation, the Tina Brozman Foundation, the Discovery to Cure Foundation, and the Guido Berlucchi Foundation (to A.D.S.). This investigation was also supported by NIH Research Grant CA-16359 from the National Cancer Institute and by Stand-up-to Cancer (SU2C) Convergence Grant 2.0 (to A.D.S.). We thank Katerina Politi (Department of Pathology, Yale University) for generous support with reagents. Preliminary results for this work were presented at the 50th Annual Meeting of the Society of Gynecologic Oncology, March 16–19, 2019, Honolulu, HI (Poster ID 1215: “Whole exome sequencing (WES) reveals novel therapeutic targets in cervical cancer”, by Lopez S. et al.). Funding Information: ACKNOWLEDGMENTS. This work was supported in part by Gilead Sciences Inc. (Foster City, CA), Puma Biotechnology, Inc. (Los Angeles, CA), NIH Grants R01 CA154460-01 and U01 CA176067-01A1, the Deborah Bunn Alley Foundation, the Tina Brozman Foundation, the Discovery to Cure Foundation, and the Guido Berlucchi Foundation (to A.D.S.). This investigation was also supported by NIH Research Grant CA-16359 from the National Cancer Institute and by Stand-up-to Cancer (SU2C) Convergence Grant 2.0 (to A.D.S.). We thank Katerina Politi (Department of Pathology, Yale University) for generous support with reagents. Preliminary results for this work were presented at the 50th Annual Meeting of the Society of Gynecologic Oncology, March 16–19, 2019, Honolulu, HI (Poster ID 1215: “Whole exome sequencing (WES) reveals novel therapeutic targets in cervical cancer”, by Lopez S. et al.). Publisher Copyright: © 2019 National Academy of Sciences. All rights reserved.
PY - 2019
Y1 - 2019
N2 - The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirustype-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/ AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.
AB - The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirustype-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/ AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.
KW - Cervical cancer
KW - Copanlisib
KW - HER2/neu
KW - Neratinib
KW - PIK3CA
UR - http://www.scopus.com/inward/record.url?scp=85074465879&partnerID=8YFLogxK
U2 - 10.1073/pnas.1911385116
DO - 10.1073/pnas.1911385116
M3 - Article
C2 - 31624127
AN - SCOPUS:85074465879
SN - 0027-8424
VL - 116
SP - 22730
EP - 22736
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 45
ER -