This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Kolev et al.
1
PI3K/mTOR dual inhibitor VS-5584 preferentially targets cancer stem cells
Vihren N. Kolev1, Quentin G. Wright1, Christian M. Vidal1, Jennifer E. Ring1, Irina M. Shapiro1, Jill Ricono2, David T. Weaver1, Mahesh V. Padval1, Jonathan A. Pachter1* and Qunli Xu1*
1Verastem Inc., Needham, Massachusetts; 2Molecular Response, San Diego, California
*Corresponding authors:
Jonathan A. Pachter, Verastem Inc., 117 Kendrick Street, Needham, MA 02494. Phone: 781-292-4215; Email: [email protected]; and Qunli Xu, Verastem Inc., 117 Kendrick Street, Needham, MA 02494. Phone: 781-292-4230; Email: [email protected]
Conflict of Interest Disclosure: V.N. Kolev, Q.G. Wright, C.M. Vidal, J.E., Ring, I.M. Shapiro, D.T. Weaver, M.V. Padval, J.A. Pachter, and Q. Xu are current or former employees of Verastem, Inc. and have equity ownership in the same. J. Ricono is an employee of Molecular Response.
Running Title: VS-5584 preferentially targets cancer stem cells
Keywords: Cancer Stem Cells, VS-5584, PI3K, mTORC1, mTORC2, breast cancer, small cell lung cancer
Abbreviations: CSC, cancer stem cells; PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; PIK3CA, catalytic subunit of PI3Kα; ALDH, aldehyde dehydrogenase; SP, side population; TIF, tumor initiating frequency; ER, estrogen receptor; SCLC, small cell lung cancer; FACS, fluorescence-activated cell sorting; MTD, maximum tolerated dose
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
APC-labeled IgG 2bK and PE labeled IgG2aK (BD Biosciences) were used as isotype controls
for CD44 and CD24, respectively.
Tumorsphere assay
To determine tumorsphere forming efficiency, cells from tissue culture or dissociated
tumors were plated in tumorsphere forming medium as previously described (17). Spheres were
enumerated using Celigo.
Apoptosis assays and siRNA transfection
Standard methods were used for Annexin V and Caspsase 3/7 assays and siRNA transfection
with details in Supplementary Materials and Methods.
In vivo Tumor xenograft studies, cell dissociation and limiting dilution assay For the SCLC PDX study: 105 cells of p2 SCLC PDXactTM tumor (Molecular Response, San Diego,
CA) admixed with Cultrex ECM (Trevigen, Gaithersburg, MD) were subcutaneously injected
into NOD-SCID mice (Harlan, Livermore CA). When tumors reached an average size of 200
mm3, mice were randomized with 5 mice per group and treated with vehicle control, cisplatin
((i.p. once a week for 2 weeks), or cisplatin followed by VS-5584 (PO, 3 times weekly for 4
weeks). Details of other tumor xenograft studies are in Supplementary Materials and Methods.
To dissociate single cells, tumor tissue was minced into smaller pieces and incubated with
Liberase (Roche Applied Science, Indianapolis, IN) or dispase for 1h at 37°C under agitation.
Mouse cells were removed from xenograft human tumors using mouse antibodies and magnetic
beads.
For the limiting dilution assay, cells dissociated from xenograft tumors were admixed with
matrigel and injected into the mammary fact pad (for MDA-MB-231 and MCF-7 breast tumors)
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
1. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645-8. 2. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine. 1997;3:730-7. 3. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:3983-8. 4. Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell stem cell. 2012;10:717-28. 5. Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell. 2009;138:822-9. 6. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nature reviews Cancer. 2008;8:755-68. 7. Varghese S, Whipple R, Martin SS, Alexander HR. Multipotent cancer stem cells derived from human malignant peritoneal mesothelioma promote tumorigenesis. PloS one. 2012;7:e52825. 8. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nature reviews Cancer. 2009;9:550-62. 9. Guertin DA, Sabatini DM. The pharmacology of mTOR inhibition. Science signaling. 2009;2:pe24. 10. Dubrovska A, Kim S, Salamone RJ, Walker JR, Maira SM, Garcia-Echeverria C, et al. The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:268-73. 11. Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006;441:475-82. 12. Zhou J, Wulfkuhle J, Zhang H, Gu P, Yang Y, Deng J, et al. Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:16158-63. 13. Korkaya H, Paulson A, Charafe-Jauffret E, Ginestier C, Brown M, Dutcher J, et al. Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling. PLoS biology. 2009;7:e1000121. 14. Yang Y, Iwanaga K, Raso MG, Wislez M, Hanna AE, Wieder ED, et al. Phosphatidylinositol 3-kinase mediates bronchioalveolar stem cell expansion in mouse models of oncogenic K-ras-induced lung cancer. PloS one. 2008;3:e2220. 15. Hart S, Novotny-Diermayr V, Goh KC, Williams M, Tan YC, Ong LC, et al. VS-5584, a novel and highly selective PI3K/mTOR kinase inhibitor for the treatment of cancer. Molecular cancer therapeutics. 2013;12:151-61. 16. Elkabets M, Vora S, Juric D, Morse N, Mino-Kenudson M, Muranen T, et al. mTORC1 Inhibition Is Required for Sensitivity to PI3K p110alpha Inhibitors in PIK3CA-Mutant Breast Cancer. Science translational medicine. 2013;5:196ra99. 17. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009;138:645-59. 18. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer research. 2005;65:6207-19. 19. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. Journal of immunological methods. 2009;347:70-8.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
20. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell. 2007;1:555-67. 21. Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer research. 2009;69:1302-13. 22. Patel S, Rameshwar P. Tumorsphere Passage for Breast Cancer Stem Cells. 2013. 23. Conley SJ, Gheordunescu E, Kakarala P, Newman B, Korkaya H, Heath AN, et al. Antiangiogenic agents increase breast cancer stem cells via the generation of tumor hypoxia. Proceedings of the National Academy of Sciences. 2012. 24. Fruman DA, Rommel C. PI3Kdelta inhibitors in cancer: rationale and serendipity merge in the clinic. Cancer discovery. 2011;1:562-72. 25. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer research. 2008;68:4311-20. 26. Kim YH, Mishima M. Second-line chemotherapy for small-cell lung cancer (SCLC). Cancer treatment reviews. 2011;37:143-50. 27. Charafe-Jauffret E, Ginestier C, Bertucci F, Cabaud O, Wicinski J, Finetti P, et al. ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program. Cancer research. 2013;73:7290-300. 28. DeRose YS, Wang G, Lin YC, Bernard PS, Buys SS, Ebbert MT, et al. Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nature medicine. 2011;17:1514-20. 29. Fang DD, Zhang CC, Gu Y, Jani JP, Cao J, Tsaparikos K, et al. Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a Mutation. PloS one. 2013;8:e67258. 30. Pinton G, Manente AG, Angeli G, Mutti L, Moro L. Perifosine as a potential novel anti-cancer agent inhibits EGFR/MET-AKT axis in malignant pleural mesothelioma. PloS one. 2012;7:e36856.
Figure Legends
Figure 1. VS-5584 preferentially targets CSC in vitro. (A) VS-5584 markedly reduced the
viability of Aldefluor+ cells but had weaker effect against Aldefluor- cells. SUM159, MCF7,
and Hs578T breast cancer cells were treated with VS-5584 for 2 days and an imaging-based
Aldefluor assay was carried out. (B) Paclitaxel and cisplatin enriched for Aldefluor+ CSC.
SUM159 cells were treated with paclitaxel or cisplatin for 2 days followed by an Aldefluor
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223
Published OnlineFirst November 28, 2014.Cancer Res Vihren N. Kolev, Quentin G Wright, Christian M Vidal, et al. stem cellsPI3K/mTOR dual inhibitor VS-5584 preferentially targets cancer
Updated version
10.1158/0008-5472.CAN-14-1223doi:
Access the most recent version of this article at:
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2014/12/02/0008-5472.CAN-14-1223To request permission to re-use all or part of this article, use this link
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 28, 2014; DOI: 10.1158/0008-5472.CAN-14-1223