Fig 2: Targeted inhibitors of DNA-PKcs or IGF1R demonstrate synergistic effect in combination with everolimus. (A–E) MM66 cells were treated with either everolimus (blue bars) or a targeted inhibitor of (A) ALK-2 (LDN-193189), (B) PRKD3 (CRT66101), (C) IGF1R (OSI-906), (D) DNA-PKcs (NU7026), (E) DNA-PKcs (KU57788) (green bars), or the combination of both (red) for 5 days to determine the effect on viability. The synergistic scores were calculated by the Excess over Bliss algorithm; they are indicated for each pair of concentrations below the X-axes (EoB). Significant reductions (p < 0.05) in viability in combinational treatment comparing to both of the single treatments are indicated with (*). Statistical analysis was performed using one-way ANOVA with Tukey’s test for multiple comparisons, and error bars present means ± SEM. (F) The effect of various concentrations of double DNA-PK/mTOR inhibitor CC-115 on viability of UM cell lines after 3 days of treatment. Significant reduction (p < 0.05) of viability comparing to a control is indicated with (*). Statistical analysis was performed using one-way ANOVA with Dunnett’s test for multiple comparisons, and error bars present means ± SEM. Every experiment was performed at least in three independent biological replicates; a representative replicate is presented.

Fig 3: PDOs and PDXs represent clinically relevant models for determination of sensitivity to DDRi in vitro and in vivo. A, Pharmacologic testing of organoids derived from patients with colorectal cancer with six different DDRi in a 7-day-long viability assay. The results at the endpoint were normalized to control wells containing DMSO vehicle and were plotted as histograms (a percent of viability) at clinically relevant concentrations of individual inhibitors. MG-132 was used as a positive control for organoid death (Supplementary Fig. S14).The screening detected strong pattern of sensitivity to ATRi, CHK1i, and WEE1i in 3 patients (#1–3). Sensitivity cutoff was set to 35% and resistance is above 75% viability (indicated with dashed lines). Unpaired Student t test was used for statistical evaluation of the results. ns, nonsignificant. B, The heatmap with indicated viability values in percentage at the end of the experiment for indicated drug concentrations. Results (A and B) are an average of at least two independent biological experiments with technical quadruplicates. Z factor values varied between 0.74 and 0.94. C, IHC detection of RAD51C and ATM protein in colorectal cancer PDOs’ cytoclots. Patient #1 displays RAD51C negativity compared with other PDOs, while all ex vivo tumor models display ATM positivity. The sections were counterstained with hematoxylin. Scale bar: 50 µm. Quantification of two independent biological experiments performed for detection of pRPA32 S33 foci at basal levels or RAD51 foci after 24-hour-long HU treatment. NT, nontreated. Statistical significance: *, P < 0.05; ns, nonsignificant (two-tailed unpaired Student t test). D, Representative images of immunofluorescence detection of RAD51 and pRPA32 S33 signal in ATRi-resistant (Patient #5) and ATRi-sensitive (Patient #1) organoids treated with 2.5 mmol/L HU for 24 hours. Scale bar: 25 µm. HU, hydroxyurea; NT, nontreated. E, ATRi-sensitive C80 cells were injected into NOD-SCID mice to establish a xenograft growth. After expansion and randomization when the tumor volume in individual mice reached 100–200 mm3, mice started being treated with ceralasertib (50 mg/kg, by oral administration daily) for 21 days. Tumor volumes were measured every 3 days using caliper. †, One mouse from the control group was sacrificed because of tumor growth reaching the endpoint. Bars, ±SE. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann–Whitney nonparametric test).

Fig 4: Effect of the DNA-PKcs and IGF1R inhibitors in combination with everolimus on UM-patient-derived zebrafish xenograft model. (A) Determination of MTD of everolimus (top left), CC-115 (top right), KU57788 (bottom left), or OSI-906 (bottom right). The zebrafish embryos were subjected to a serial dilution of the inhibitor. Treatments were refreshed every other day for a total treatment duration of 6 days. The MTD was determined as the concentration where at least 80% of the treated embryos survived the treatment. (B) Effect of everolimus (1.25 µM), OSI-906 (1.25 µM), KU-57788 (0.3125 µM), and CC-115 (10 µM) alone or combined on tumor burden in zf-PDX UM model. Asterisk (*) indicates significant reduction (p < 0.05) compared to a control. Statistical analysis was performed using one-way ANOVA with Dunnett’s test for multiple comparisons.

Fig 5: CCL5-CCR5 paracrine signaling activates DDR signaling in GBM cells to potentiate TMZ resistance.a Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells were stimulated with CCL5 (10 ng/mL) or vehicle (PBS) followed by the administration of TMZ (500 μmol/L) or DMSO. ns, not significant. **P < 0.01. b Cell survival analysis of GBM-1 cells with indicated treatments. CM from pericytes expressing shNT or shCCL5 (sh-1 or sh-2) was collected and added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after CM addition. **P < 0.01. c Cell survival analysis of GBM-1 cells with indicated treatments. Pericyte CM was pretreated with neutralizing anti-CCL5 antibody or IgG before being added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after pericyte CM addition. **P < 0.01. d Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells expressing shNT or shCCR5 (sh-1 or sh-2) were pretreated with pericyte CM or control GBM cell CM followed by TMZ (500 μmol/L) treatment. *P < 0.05; **P < 0.01. e Schematic diagram of MVC-mediated inhibition of CCL5-CCR5 signaling. The binding of MVC to CCR5 leads to a conformational change of its extracellular domain to prevent ligand-stimulated CCR5 activation. f Apoptosis analysis of GBM-1 cells with indicated treatments. GBM-1 cells were pretreated with MVC (500 nmol/L) or DMSO for 1 h, followed by pericyte CM stimulation. Apoptosis and cell survival analyses were performed 48 h after TMZ treatment. ns, not significant. **P < 0.01. g DNA damage after the indicated treatments was assessed by comet assay. Scale bars, 50 μm. h Quantification of percentage of cells with comet tails with indicated treatments. ns, not significant. *P < 0.05; **P < 0.01. i, j Immunoblot analysis of phosphorylated AKT (Ser473) and AKT (i) or phosphorylated DNA-PKcs (Ser2056) and DNA-PKcs (j) in GBM-1 cells with indicated treatments. k Immunoblot analysis of phosphorylated AKT (Ser473), AKT, phosphorylated DNA-PKcs (Ser2056), DNA-PKcs, γ-H2AX in GBM-1 cells with indicated treatments. DNA-PKi (DNA-PKcs inhibitor) represents KU-57788; AKTi (AKT inhibitor) represents MK-2206. Experiments in a–k were independently performed three times.