Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A Romi Guptaa, Yuying Donga, Peter D. Solomona, Hiromi I. Wetterstenb, Christopher J. Chengc,d, JIn-Na Mina,e, Jeremy Hensonf,g, Shaillay Kumar Dograh, Sung H. Hwangi, Bruce D. Hammocki, Lihua J. Zhuj, Roger R. Reddelf,g, W. Mark Saltzmanc, Robert H. Weissb,k, Sandy Changa,e, Michael R. Greenl,1, and Narendra Wajapeyeea,1 Departments of aPathology and eLaboratory Medicine, Yale University School of Medicine, New Haven, CT 06510; iDepartment of Entomology and bDivision of Nephrology, Department of Internal Medicine, University of California, Davis, California 95616; Departments of cBiomedical Engineering and dMolecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511; fSydney Medical School, University of Sydney, NSW 2006, Australia; gCancer Research Unit, Children’s Medical Research Institute, Westmead, NSW 2145, Australia; hSingapore Institute of Clinical Sciences, Agency for Science Technology and Research (A*STAR), Brenner Center for Molecular Medicine, Singapore 117609; lHoward Hughes Medical Institute and jPrograms in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Massachusetts 01605; and kDepartment of Medicine, Mather VA Medical Center, Sacramento, CA 9565 Contributed by Michael R. Green, June 19, 2014 (sent for review June 8, 2014) Tumor suppressor p53 plays an important role in mediating growth dition to its role in cell cycle regulation, p21 has been shown in inhibition upon telomere dysfunction. Here, we show that loss of a variety of studies to repress apoptosis (9–13). the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, Here,westudytheroleofp21inthe context of telomerase in- also known as p21WAF1/CIP1) increases apoptosis induction following hibition. We find that abrogation of p21 function induces apoptosis telomerase inhibition in a variety of cancer cell lines and mouse in cancer cells following telomerase inhibition through up-regula- xenografts. This effect is highly specific to p21, as loss of other tion of p53-upregulated modulator of apoptosis (PUMA), a proa- checkpoint proteins and CDK inhibitors did not affect apoptosis. In poptotic protein. Based upon these results, we go on to show that telomerase, inhibited cell loss of p21 leads to E2F1- and p53-medi- simultaneous genetic or pharmacological inhibition of telomerase ated transcriptional activation of p53-upregulated modulator of and p21 can synergistically suppress tumor growth, even in p53 apoptosis, resulting in increased apoptosis. Combined genetic or pathway-defective cancers. pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simul- Results taneous inhibition of telomerase and p21 also suppresses growth of Induction of Apoptosis Following Telomerase Inhibition in Cancer Cells tumors containing mutant p53 following pharmacological restora- Lacking p21. As described above, p53 is known to play an impor- tion of p53 activity. Collectively, our results establish that inacti- tant role in the cellular response to telomere dysfunction, and vation of p21 leads to increased apoptosis upon telomerase p21 is a major target of p53. However, the specific role of p21 in inhibition and thus identify a genetic vulnerability that can be human cancer cells with dysfunctional telomeres has not been exploited to treat many human cancers containing either wild-type examined. Therefore, we asked whether cancer cells respond or mutant p53. differently to telomerase inhibition and consequential telomere shortening in the presence or absence of p21. Toward this end, we prominent feature that distinguishes cancer cells from their treated HCT116 cells and HCT116 p21 knockout cells (HCT116 Anormal counterparts is the expression of telomerase. Telo- p21KO) with the telomerase inhibitor imetelstat (14). We found merase is a specialized ribonucleoprotein reverse transcriptase that imetelstat inhibited proliferation of HCT116 p21KO cells that synthesizes the telomeric DNA ends to maintain telomere length (1). During early tumorigenesis, telomerase expression is Significance necessary to bypass replicative senescence, enabling immortali- zation of human cells (2). Notably, telomerase also represents an Over 90% of cancer cells express telomerase, which is required attractive target for cancer therapy because a large majority of for their survival. However, telomerase inhibitors alone have cancer cells depend on telomerase expression for survival. Ac- so far failed to provide any significant clinical benefit. There- cordingly, genetic or pharmacological inhibition of telomerase fore, identifying and targeting genes that can enhance the has been shown to suppress growth of cancer cells (3). In fact, effects of telomerase inhibitors will greatly benefit a large the telomerase inhibitor imetelstat, an oligonucleotide that in- population of cancer patients. We find that simultaneous in- hibits telomerase activity by binding to the RNA component of hibition of p21 and telomerase synergistically suppresses tu- human telomerase RNA (hTR), has advanced to the clinic for mor growth. We also show that our approach is useful for treatment of various hematological malignancies and solid treating p53 mutant cancers, when used with therapies that tumors. To date, however, telomerase-based monotherapies restore the function of mutant p53. We anticipate that simul- have not been successful, underscoring the need to understand taneous targeting of p21 and telomerase will overcome the in greater detail how cancer cells respond to telomerase inhibition. current limitation of single-agent telomerase therapeutics and Previous studies have shown that the tumor suppressor p53 provide an effective method to treat cancers that rely on telomerase activity for survival. pathway plays a central role in regulating the cellular response to telomerase inhibition and telomere shortening (4). Genetic de- Author contributions: R.G. and N.W. designed research; R.G., Y.D., P.D.S., C.J.C., J.-N.M., letion of p53 in mice or RNA interference (RNAi)-mediated and N.W. performed research; R.G., H.I.W., C.J.C., J.H., S.K.D., S.H.H., B.D.H., R.R.R., W.M.S., inhibition of p53 counteracts the growth suppression that occurs R.H.W., S.C., M.R.G., and N.W. contributed new reagents/analytic tools; R.G., Y.D., P.D.S., S.K.D., L.J.Z., S.C., M.R.G., and N.W. analyzed data; and R.G., M.R.G., and N.W. wrote following telomerase inhibition. Furthermore, p53 loss cooper- the paper. ates with telomerase dysfunction to promote tumorigenesis (4). The authors declare no conflict of interest. CDKN1A (also known as p21WAF1/CIP1) is a cyclin-dependent ki- 1To whom correspondence may be addressed. Email: [email protected] or nase inhibitor and a direct transcriptional target of p53 (5, 6). p21 [email protected]. mediates several important physiological effects of p53, includ- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ing DNA-damage–induced cell cycle checkpoints (7, 8). In ad- 1073/pnas.1411370111/-/DCSupplemental. E3062–E3071 | PNAS | Published online July 14, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1411370111 Downloaded by guest on September 23, 2021 PNAS PLUS *** *** 120 Sub G1 S A HCT116 B C D G1 G2/M 100 Wild type p21KO 100 100 80 80 80 60 60 60 Mismatch 40 40 40 % Cell population 20 20 Cell viability (%) 20 0 0 activity (%) Telomerase 0 Imetelstat - + - + Imetelstat -+ -+ Imetelstat -+ -+ Imetelstat Wild type p21KO Wild type p21KO Wild type p21KO HCT116 1500 E F 50 *** G Mismatch: ) Wild type 3 40 1200 p21KO Wild type p21KO Imetelstat: - + Imetelstat 30 -+ 900 Wild type *** Cleaved Caspase 3 20 p21KO 600 Actin 10 300 Tumor volume (mm Tumor % Annexin V positive cells % 0 Imetelstat -+ -+ Wild type p21KO 0 7 14 21 28 Days Fig. 1. Telomerase inhibition induces apoptosis in the absence of p21. Indicated cell lines were treated with mismatch oligonucleotide or imetelstat for 6 wk. (A) Crystal violet staining of HCT116 wild-type and p21KO colonies. Representative wells are shown. (B) Relative cell viability was monitored by trypan blue exclusion assay. (C) Telomerase activities of HCT116 wild-type and HCT116 p21KO cells. (D) Flow cytometry analysis to monitor apoptotic cells. (E) Cleaved caspase 3 immunoblot to measure apoptosis. Actin was used as a loading control. (F) % Annexin V–FITC–positive cells under indicated treatment conditions. (G) Average tumor volumes for indicated cell lines are presented at indicated conditions. ***P < 0.0001. much more strongly than that of HCT116 cells (Fig. 1 A and B). and UACC62 cells was higher than that of the mismatch oligo- Notably, telomerase inhibition in HCT116 and HCT116 p21KO nucleotide-treated cells (SI Appendix, Fig. S3), possibly due to cells was comparable (Fig. 1C). Additional experiments revealed the activation of Alternative Lengthening of Telomeres (ALT) that growth inhibition of HCT116 p21KO cells was largely due to pathway (SI Appendix,Fig.S3). Taken together, these results increased apoptosis (Fig. 1 D–F). Furthermore, we knocked down indicate that loss of p21 sensitizes diverse cancer cell lines to telomerase using two different short hairpin RNAs (shRNAs) in tumor inhibition and apoptosis following abrogation of telo- HCT116 and HCT116 p21KO cells. Similar to the results with merase activity. imetelstat, we found that shRNA-mediated knockdown of telo- merase inhibited proliferation of HCT116