Telomeres protect the ends of linear chromosomes from being recognized as damaged DNA, and telomere stability is required for genome stability. Here we demonstrate that telomere stability in androgen receptor (AR)-positive LNCaP human prostate cancer cells is dependent on AR and androgen, as AR inactivation by AR antagonist bicalutamide (Casodex), AR-knockdown, or androgen-depletion caused telomere dysfunction, and the effect of androgen-depletion or Casodex was blocked by the addition of androgen. Notably, neither actinomycin D nor cycloheximide blocked the DNA damage response to Casodex, indicating that the role of AR in telomere stability is independent of its role in transcription. We also demonstrate that AR is a component of telomeres, as AR-bound chromatin contains telomeric DNA, and telomeric chromatin contains AR. Importantly, AR inactivation by Casodex caused telomere aberrations, including multiple abnormal telomere signals, remindful of a fragile telomere phenotype that has been described previously to result from defective telomere DNA replication. We suggest that AR plays an important role in telomere stability and replication of telomere DNA in prostate cancer cells, and that AR inactivation-mediated telomere dysfunction may contribute to genomic instability and progression of prostate cancer cells.

Background: Androgen receptor (AR) inactivation causes telomere dysfunction. Results: AR-inactivation-induced telomere dysfunction led to the activation of ATM at telomeres, and ATM inhibition blocked repair of damaged telomeric DNA and augmented cell death. Conclusion: ATM promotes survival of AR-inactivated prostate cancer cells with telomere dysfunction. Significance: ATM inhibitors may potentiate the efficacy of AR-targeted therapies for the treatment of prostate cancer.

Telomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant.

Prostate cancer is the most commonly diagnosed non-skin cancer, and second leading cause of cancer deaths, in American men. The androgen receptor (AR) plays a critical role in the survival of prostate cancer (PCa) cells. Therefore, treatments that inactivate AR, either by decreasing the androgen level or by inhibiting AR directly, are used to treat advanced PCa. However, these treatments are not curative, and disease progression generates castration-resistant prostate cancer (CRPC). Our studies point to a novel role of AR in telomere stability that, in our opinion, can be exploited to enhance the effectiveness of AR-targeted therapies for CRPC. Telomere stability is essential for cell proliferation and survival. We previously reported that AR plays a role in maintaining telomere stability in prostate cancer cells. Since telomere dysfunction activates the DNA damage response (DDR) signaling pathway which can activate cell cycle checkpoints, repair damaged DNA, and promote cell survival, we tested a hypothesis that AR-inactivated telomere dysfunction activates a DDR signaling pathway protein, called ATM (ataxia telangiectasia mutated) kinase, and ATM inhibitor potentiates cell death by averting telomeric DNA repair in AR-inactivated PCa cells. ATM pathway was assessed by the phosphorylation of ATM and its downstream target Chk2 through Western blotting, inhibition of telomere DNA repair was evaluated by counting RPA (DNA replication protein A) foci at damaged telomeres, and the cell viability was determined by PARP cleavage and colony formation assays. We observed that telomere dysfunction by AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation of ATM and Chk2 and the presence of phosphorylated ATM at telomeres, indicating the activation of DNA damage response signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation and induced PARP cleavage, suggesting that ATM inhibitors induce apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomere. This was further corroborated by a conspicuous localization of RPA at damaged telomeres; presence of RPA foci is indicative of unrepaired stretch of single-stranded DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration- resistant prostate cancer. Citation Format: Sahn-Ho Kim, Vidyavathi Reddy, Min Wu, Evelyn R. Barrack, G. Prem-Veer Reddy. ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1815.

Telomere stability is important for cell viability, as cells with telomere DNA damage that 24 is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist 25 induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers 26 a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking 27 ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR 28 antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 29 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR 30 antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself 31 had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi 32 significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA 33 damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell 34 death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice 35 variant AR-V7, but full-length AR was found to be the predominant form of AR associated with 36 telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells 37 appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them 38 sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this 39 combined treatment approach to induce telomere DNA damage and inhibit the DDR was 40 effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the 41 use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging 42 disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express 43 AR splice variant. 44 45 3 46 Introduction: 47The critical role of the androgen receptor (AR) in prostate cancer cell proliferation and 48 survival is the enduring basis for treating advanced prostate cancer with drugs that block AR 49 function or androgen biosynthesis (1, 2). However, a relentless challenge is the development of 50 resistance to these treatments, referred to as castration-resistant prostate cancer (CRPC) (3). 51Remarkably, CRPC still relies on AR (4, 5), indicating a need to more fully understand the role 52 of AR in cell survival. In this regard, we have discovered a role of AR in prostate cancer cell 53 telomere stability (6, 7). Notably, inactivation of this role of AR creates a DNA damage 54 response (DDR) target, inactivation of which blocks repair and promotes cell death (8). 55Telomeres are the DNA-protein structures that cap the ends of linear chromosomes, 56 which are double-stranded DNA with a single-stranded overhang (9). Telomeres contain many 57 different proteins that play a role in the maintenance of telomere stability; the best characterized 58 are the six proteins (TRF1, TRF2, Rap1, TIN2, POT1 and TPP1) that comprise a complex 5...