Figure 4 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

<p>Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability. <b>A,</b> Chemical structures of the Polθ inhibitors ART558 and ART899. The table shows the <i>in vitro</i> intrinsic clearance values of ART558 and ART899 after exposure to rat and mouse liver microsomes. <b>B,</b> Nano-luciferase MMEJ assay showing ART899-mediated inhibition of MMEJ activity in HEK-293 cells. The nano-luciferase readings were normalized to control luciferase (firefly) readings, and these were then normalized to DMSO. Data points show the mean ± SEM of four technical replicates; representative of two independent experiments. <b>C,</b> Confirmation of MMEJ assay specificity. Same experiment described in <b>B</b> but showing both the nanoluc and firefly readings normalized to their own DMSO reading, confirming negligible inhibition by ART899 of the control firefly luciferase signal. <b>D,</b> Clonogenic survival of HCT116 and H460 cells treated with ART899. Graphs show the surviving fraction after 5 × 2 Gy IR. <b>E,</b> Confirmation of ART899 specificity in U2OS WT and Polθ KO cells. Cells were treated as described in <b>D</b>. <b>F,</b> Effect of ART899 in noncancerous cells. MRC-5 and AG01552 fibroblasts were treated as described in <b>D</b>. The effect of ART899 in unirradiated cells from <b>D</b> to <b>F</b> is shown in Supplementary Fig. S5A. Graphs shown in <b>D</b> to <b>F</b> correspond to average ± SD from triplicate wells (representative from three separate experiments; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001). <b>G,</b> Viability of HIEC-6 cells treated with ART899 and irradiated with 5 × 2 Gy, as determined by the alamar blue assay 8 days after the first IR fraction. Graph shows the viability normalized to unirradiated controls (Supplementary Fig. S5C); representative from three independent experiments.</p>