Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy

Abstract

The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment.

Keywords: DNA damage response; inhibitors of Tousled kinases; mechanism of DSB repair; radiomimetic sensitizers.

Figure 1.

Inhibition of TLK1/1B autophosphorylation by phenothiazines. (A) Selected phenothiazines were tested in autokinase assays employing γ³²P-ATP and determined by TCA-precipitable counts (% inhibition compared to no drug). (B) The TLK1/1B inhibitors were tested in cultured cells by IP/autokinase/autoradiography. 293T cells were incubated for 1 hour with TLK1/1B inhibitors (TFP and PPH). Extracts were prepared for IP as described in Materials and Methods, followed by autophosphorylation with γ³²P-ATP. The blots were then probed with TLK1 antiserum.

Figure 2.

Kinetics of repair in episomes. (A) Kinetics of repair in episomes cleaved with adeno-HO endonuclease (Ad-HO) during a time course of infection. 293T cells were treated (or not) with TFP or PPH 12 hours prior to infection. Kinetics of repair was followed by qPCR with primers flanking the HO cut site, at the pRSV promoter and at the CAT gene. Data points were normalized for an amplicon in the Amp gene and also for a genomic site to ensure the isolation of equal amounts of DNA. (B) Infection with Ad-HO and generation of a single DSB in episomes result in the phosphorylation of ATM (activation), TLK1(S695) (inhibition), and H2AX(S139). Infection of 293T cells that do not contain the HO-targeted episomes does not result in sufficient ATM activation (bottom panel).

Figure 3.

Presence of DSB repair foci (γ-H2AX foci) after DSB induction in DU145 cells. (A) DSBs were induced by 3 µM of Bleocin (Calbiochem, San Diego, CA [Cat. No. 203408]). Cells were treated with 3 µM Bleocin and +/- 10 µM TFP for 24 hours. After 24 hours cells were washed with 1XPBS and fresh media was added. Cells were allowed to recover for 0, 2, and 4 hours respectively at 37°C. γ-H2AX (red) signal was captured using a Zeiss Axioskop microscope after 0, 2 and 4 hours of recovery time. (B) Percentage of cells with ≥the indicated number of γ-H2AX foci, at least 150 cells per treatment, and time point were counted and grouped into tertiles; error bars were not added since this analysis is largely qualitative, unless thousands of cells can be accurately scored.

Figure 4.

Repair of a DSB in vitro: ligation of EcoRI/EcoRV-cut plasmid. The effect of TFP on incompatible end filling and ligation/supercoiling and Rad9 supplementation. The top and bottom panels (EtBr and autorad) show the time course of plasmid religation/supercoiling, which includes filling of the EcoRI site with radiolabeled dATP (5 μCi [α-³²P] dATP/0.1 mM) by endogenous polymerases. Ligation of the ends and assembly of chromatin are shown by the accumulation of faster mobility topoisomeric forms; these are the result of nucleosome-driven formation of negative supercoils.^, The EtBr band of highest compaction (s.c.II) was graphically quantitated below each lane with ImageJ software (National Institutes of Health).

Figure 5.

The TLK inhibitors block the phosphorylation of Rad9(S328) and impair the deactivation of the DDR. (A-C) DU145 cells were incubated with TLK inhibitors (PPH-LS125 or THD-LS078) for 2 hours and then with H₂O₂. A sample without H₂O₂ DSB induction is shown in lanes 8 and 12. (D) Chk1 remains activated (phosphorylated) after the recovery from H₂O₂ in cells treated with inhibitors. (E) Rad17(S645) remains phosphorylated in the presence of LS125 and LS078, which alone do not result in Rad17 phosphorylation (not shown). (F) Cleavage of CDC25A and release of the 33-kDa product, consequent to Chk1 activation, which are expected to result in a quicker and prolonged cell cycle arrest (as was shown in Suppl. Fig. S2).

Figure 6.

TLK inhibitors potentiate RMT killing in DU145 PC-3 and MDA-MB231–Luc cells. (A, B) Viability of DU145 and PC-3 cells with varying concentrations of doxo + TFP, THD, or PPH (10 µM) added 12 hours before the addition of doxo.(C, D) Viability with various concentrations of phenothiazines only. Viability was measured after 24 hours with CellTiter 96 AQueous One Solution Cell Proliferation (Promega). (E) Viability of MDA-231–Luc BCA cells. Treated and control cells were assessed with luminescence. RLU values were measured, with the Promega Luciferase Kit, as a direct viability indicator with DSBs induced with increasing Bleocin (Calbiochem, San Diego, CA [Cat. No. 203408])..

Figure 7.

Tumor growth in mice. Xenograft assays (10⁶ cells injected s.c.) were used to assess the effect of THD and doxo in the growth of CaP tumors, PC-3, in male mice or MDA-MB231–Luc in females. Tumor size was monitored twice weekly with calipers and by in vivo imaging. Groups included control, THD and doxo, and THD + doxo.