Abstract 2821: Tousled like Kinase 1 binds to and regulates the protein kinase Nek1: Its implication in prostate cancer

Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR). Through a novel proteomic approach, we have identified that NIMA kinase NEK1 strongly interacts and co-localizes with TLKs and has a role in the DDR, upstream of ATR and Chk1. In the present study we have tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in prostate cancer cell lines and LNCaP cell derived xenografts in NOD/SCID mice and the subsequent role of ATR-Chk1 axis following DNA damage. Material & Methods: We have used a prostate cancer cell line viz. LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1. Clonogenic activity was measured by colony formation assay. Immunoblotting was done for TLKs, NEK1, p-ATR and p-Chk1 protein in PCa cell line and tumor xenografts tissues. LNCaP xenografts in NOD/SCID mice were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model. Results: Treatment with THD suppresses the outgrowth of androgen-independent (AI) colonies of LNCaP cells cultured with bicalutamide. Moreover, THD significantly inhibited the colony formation efficiency of several PCa cells in vitro (even of androgen independent lines). Intra-Peritoneal (I.P.) administration of THD or Bicalutamide long-term was not very effective in inhibiting tumor growth. In contrast, combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide as evident by immunoblotting of residual tumors. In cells, following DNA damage, addition of the TLK inhibitor THD impaired ATR and Chk1 activation, indicating the existence of a TLK1>NEK1>ATR>Chk1 pathway. Conclusions: Our data indicated that THD repressed growth of PCa cells in vitro and in vivo, and for Androgen-dependent (LNCaP) cells, in combination with bicalutamide it was very effective at preventing outgrowth of AI cells and hence as a targeted ADT adjuvant therapy. Moreover, THD impaired DDR. Collectively, these results strongly suggest that THD might be a novel anti-tumor agent for use in prostate cancer. Citation Format: Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. Tousled like Kinase 1 binds to and regulates the protein kinase Nek1: Its implication in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2821.