DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2β-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

www.nature.com/scientificreports/ 3D spheroid 96-well assay. When spheroids reached a diameter of ~ 400 µm they were collected and dispensed into 2 mL tall polypropylene 96-well plates (Axygen). Settled spheroids were dispensed in 3-5 µL volumes into 1.2 mL of drug containing media to achieve 5-10 spheroids per well using a wide bore tip (Perkin Elmer, wide bore-P235) with an automated 96-channel liquid handler (PerkinElmer, Evolution P3). Multi-well plates were set up with desired drug dilutions prior to adding spheroids. Plates were then sealed with a silicone rubber mat (Axygen) in a glove box (Hypoxygen) at controlled gassing of 5% CO 2 , 20% O 2 . Upon sealing plates, they were then continuously rotated (20 rpm) with plates oriented on their sides and tilted so as to use the trapped air bubble to mix and maintain the spheroids in a free-falling state. Spheroids were exposed to etoposide ± inhibitors for up to 24 h prior to collection for cryo-sectioning or cell survival endpoints.
3D spheroid 96-well immunohistochemical endpoint. Following treatment as indicated, spheroids were incubated for 2 h with either 100 µM 5-ethynyl-2′-deoxyuridine (EdU) (Gold biotechnology) for replication or 1000 µM 5-ethynyluridine (EU) (Carbosyth China Ltd), for transcription prior to collection for cryosectioning and click-based staining. Spheroids were collected from all 96-wells simultaneously using a 96-channel pipetting head, allowed to settle and then dispensed as liquid-free piles on a stretched 13 × 13 cm latex sheet (Rite Dent) such that when relaxed the area contracted from the size of a 96-well plate (7.5 × 13 cm) down to a microscope slide (~ 2.5 × ~ 3.8 cm). The resulting spheroid micro-arrays were then immediately frozen via a -30 °C aluminium block placed under the relaxed latex sheet and embedded in OCT sectioning medium (Fisher Scientific).
3D spheroid clonogenic endpoint. Following treatment as indicated, spheroids were collected and transferred to fresh drug-free media. Spheroids were washed three times with PBS prior to 20-min incubation with 0.25% trypsin-EDTA (Gibco) with continuous agitation at 37 °C. Following dissociation, cell suspensions were further diluted in fresh media with 10% FBS. Cell suspensions were then plated using either 6 cm tissue culture plates (Sarstedt, Germany) for experiments producing greater than 90% cell kill, or clear flat bottom 96-well polystyrene cell culture plates (Corning, Costar 3595) for low cell kill experiments. The 96-well colony plates were then left for 45 min prior to placement in 37 °C humidified incubator at 5% CO 2 to allow settling of cells, otherwise non-uniform colony clumping in outer wells would occur. Following colony plating, the original cell suspensions were then counted in duplicate by diluting 25 µl aliquots from each well into additional flat-bottom clear polystyrene 96-well plates containing 25 µl of Hoechst 33342 (10 µl/ml final concentration) and propidium iodide (4 µl/ml final concentration). Cell suspensions were allowed to settle for 30 min prior to imaging using a fluorescence microscope for initial live/dead cell counting. Cell counts were then used to calculate the actual number of cells plated in individual wells. Counts were performed in replicates of 2 to ensure reproducibility. 96-well plates were incubated for 4 days at which point they were fixed in 1% formalin and stained with Hoechst 33342 for 2 h and images of each well were then taken by fluorescence microscope at 4× magnification and colonies were counted using Image J software with custom defined algorithms. 6 cm plates were incubated for 12 days to allow colony formation and were then stained using 2 g/L malachite green (Sigma-Aldrich) in water and counted manually.
In vivo studies. All studies involving mice were performed at BC Cancer Research Institute Animal Resource Centre and were approved by the University of British Columbia Institutional Animal Care and Use Committee under approved animal study protocol A21-0059. All animals were treated in accordance with the Canadian Council on Animal Care ethical guidelines and are reported in accordance with ARRIVE 2.0 guidelines. Rag2M mice were originally purchased from the Taconic Biosciences. For A549 tumor growth delay studies, 10 6 A549 cells were inoculated in the gastrocnemius muscle of 61 10-13-week-old Rag2M male mice in a volume of 50 μL, including an overage of 20% to account for variable tumor growth rates. When the median tumor volume of the cohort exceeded 150 mm 3 animals were stratified based on tumor volume ranges; animals bearing the smallest tumor volumes were excluded from the study (n = 13). Remaining animals (cohort size of n = 48, 30 for etoposide tumor growth delay and 18 for PLD growth delay) all had tumor volumes within the volume range of 160-225 mm 3 . Animals were assigned to treatment groups using stratified randomization and a random number generator to ensure a range of tumor volumes was assigned to each group. No power calculations were performed a priori. A sample group size of n = 6 was determined appropriate for this study due to prior observations of consistent tumor response to chemotherapy and sensitization responses by DNA damage repair inhibitors, without numerous outliers or abnormalities. Significant morbidities were observed that required humane termination endpoints due to significant weight loss in particular treatment groups; larger group sample sizes would not have improved this result.
Etoposide was administered intraperitoneally (ip) at 5 mg/kg on days 0, 1 and 2 of each week for 3 weeks. Pegylated liposomal doxorubicin (PLD) was administered by intravenous (iv) injection at 6 mg/kg once per week for 3 weeks. AZD7648 was administered via oral gavage (po) at 10, 30 or 100 mg/kg at the same times in combination with Top2 poisons. For control groups, an equivalent volume of drug vehicle was administered. Tumor size and mice weights were monitored 3 times per week, with tumor volumes measured by calipers and calculated using formula V = π/6 (L × H × W). Measurements were done via a blinded recorder. Mice were euthanized at experimental end point, when tumor volumes reached 1000 mm 3 , or at humane end point, including animals exceeding > 20% loss from pre-treatment weights. Animals that had to be euthanized due to humane endpoint are reported in the study results. All animals were maintained in the same housing and husbandry conditions, with dough diet supplements provided to all experimental mice during the treatment period.
For immunohistochemistry-based studies, a total of 18 mice were used. No power calculations were performed a priori. A sample size of n = 6 was determined appropriate for this study due to prior observations of Image acquisition and data analysis. Slides were loaded onto a custom-built imaging system a Nikon Plan Fluorite Imaging 10× objective (Nikon), a cooled PCO Edge 4.2 sCMOS camera (PCO), run using customized algorithms in ImageJ software (ImageJ, https:// imagej. nih. gov/ ij/). Using this system, images of entire microscope slides were captured at a resolution of 0.65 µm/pixel. Using the ImageJ software application and user supplied algorithms, images of CD31 fluorescence, 53BP1, EdU and Hoechst 33342 from each tumor section were overlaid, tumor boundaries described, and areas of necrosis and staining artefacts were removed (folds, tears, debris, etc.). Objects positive for CD31 were then identified on the CD31 image layer using a threshold determined to be 10 standard deviations above the tissue background levels. CD31 objects that were less than 10 µm 2 in size were considered artefacts and removed from the analysis. Analysis was then carried out to measure the distance from each point in the tissue to the nearest CD31 positive object noting its intensity to then determine the relation between proliferation or 53BP1 signal as a function of the distance to the nearest blood vessel. The data were tabulated so as to determine the profile of EdU and 53BP1 staining versus distance to blood vessels, or the average intensity of pixels is reported for p53BP1. A similar approach was taken for spheroid analysis, using the outer spheroid edge as the reference point rather than the CD31 objects.
Statistical analysis. All statistical analyses were performed using Graphpad Prism software (version 9.3).
Drug dose response survival curves were fitted with non-linear regression curve fitting tools in Prism, using the "Absolute IC 50 , X is log (concentration)" model. Survival summary and etoposide pIC 50 bar graphs were assessed via unpaired, two tailed t-tests. Time to tumor volume tripling was assessed for all groups by ANOVA followed by Turkey multiple comparisons tests. Mouse percentage weight loss at day 7/14 analyses were assessed by ANOVA Kruskal-Wallis test. Tumor p53BP1 signal intensity analyses were performed via unpaired two tailed t-tests. Spheroid p53BP1 signal intensity analyses were via ANOVA followed by Tukey multiple comparisons tests. Calculated p-values are reported as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; ****p ≤ 0.001.

Results
Abrogation of the NHEJ repair pathway but not HR sensitizes 3D tumor spheroids to etoposide. 3D spheroid tissue models re-capitulate the oxygen and nutrient gradients of the solid tumor microenvironment to result in a mixed cell population containing a greater proportion of non-proliferating quiescent cells relative to monolayer culture, where proliferating cells are more likely to dominate response to etoposide treatment 17 . In the 3D spheroid model, treatment with etoposide has a dose-response relationship that saturates beyond about 10 µM, with approximately 40% of cells surviving even at 10× higher doses (Fig. 1). To elucidate the roles of HR and NHEJ in sensitizing the spheroids to Top2 poison induced DNA damage, we performed clonogenic survival assays on HCT-116 spheroids that were either deficient in HR (BRCA2 −/− ) or NHEJ (PRKDC −/− ) following etoposide exposure. After 24 h, HR deficient spheroids show a similar etoposide sensitivity profile relative to wild-type HCT-116 spheroids, with approximately 60% cell kill, consistent with selective targeting of the s-phase fraction within spheroids 18 , while NHEJ-deficient spheroids experienced ~ 4 logs additional cell kill. The addition of 1 µM DNA-PK inhibitor M3814 increased the cell kill in wild-type spheroids by a factor of 10 compared to etoposide treatment alone. These results indicate that NHEJ is the main repair pathway for etoposide induced DNA damage and DNA-PK inhibition can effectively sensitize cell spheroids beyond what increasing concentrations of etoposide alone is able to achieve.

DNA-PK inhibitor sensitizes non-proliferating cells to etoposide treatment in monolayer culture.
The effect of DNA-PK inhibition in combination with Top2 poisons was assessed via clonogenic survival assays performed on monolayer cell cultures treated with etoposide ± DNA-PK inhibitors AZD7648 or M3814. Dose response curves for FaDu cells ( Fig. 2A left panel) show monotherapy treatment with AZD7648 or M3814 had no effect on survival up to 10 µM, however 1 µM of either DNA-PK inhibitor in combination www.nature.com/scientificreports/ with etoposide showed a significant reduction in cell survival relative to etoposide alone. These data were used to calculate etoposide IC 50 values shown in Fig. 2A (right panel) for FaDu and additional cell lines, with values consistently lower for 1 µM AZD7648 + etoposide relative to the Top2 poison alone, indicating that DNA-PK inhibitors sensitize monolayer tumor cells to etoposide by at least a factor of 10.
The amount of DNA damage sustained by cells in G 1 , S and G 2 stages of the cell cycle was assessed using flow cytometry based on propidium iodide (PI) staining and analysis of p53BP1 staining following treatment with etoposide ± DNA-PK inhibitor AZD7648. Results in A549 cells (Fig. 2B) show an increase in p53BP1 signal intensity for cells co-treated with etoposide + AZD7648 (78.4% of cells are positive) relative to etoposide alone (55% of cells are positive). Cell cycle-specific analysis indicates a significant increase in DNA damage for the combination relative to etoposide alone for the G 1 cell population, with smaller changes in the proliferating S and G 2 cell populations. Similar results are seen with 48BR (human skin fibroblasts) cells where these normal cells sustained significantly more DNA damage when treated with etoposide + AZD7648 compared to etoposide alone, with the largest effects in G 1 phase (Fig. 2C).
The impact of etoposide ± DNA-PK inhibition on non-proliferating cells was assessed using a clonogenic survival assay on serum-starved cells. Monolayer FaDu, SiHa, HCT-116 and A549 cells were incubated in serum-free media for 24h to result in over 70% of cells residing in G 0/1 phase (FaDu cell line shown in Fig. 2D). Clonogenic survival assays were then performed on both serum-starved and non-starved cells to generate dose-response curves for etoposide (2.5 nM-50 µM) ± 1 µM AZD7648 for 24 h. Results show that serum-starved cells are more resistant to etoposide treatment than the non-starved cells, however the addition of DNA-PK inhibitor AZD7648 re-sensitized the serum-starved cells (Fig. 2E left panel) to a similar degree as was seen in the non-serum starved cells ( Fig. 2A left panel). These results were repeated in multiple cell lines, where the IC 50 value of etoposide alone is consistently much higher than etoposide in combination with AZD7648 ( Fig. 2E right panel).

DNA-PK inhibitors sensitize non-proliferating, transcriptionally active cells in 3D tumor spheroids to etoposide.
To further explore the sensitization of cells to Top2 poisons by DNA-PK inhibition, we evaluated this combination therapy in tumor spheroids. As described above, 3D tissue spheroid models contain a greater proportion of non-proliferating, quiescent cells relative to monolayer culture. Figure 3A shows a dose-response survival curve of FaDu spheroids to etoposide, indicating the spheroids are highly resistant to etoposide compared to monolayer culture, however addition of 1 µM M3814 shows sensitization beyond the maximum effects seen with etoposide alone. Similar results are shown in additional cell lines HCT-116, SiHa, and A549, where the surviving fraction of spheroids decreased consistently in the combination treatment groups of 25 µM etoposide + 1 µM M3814 relative to 25 µM etoposide alone.
To investigate the potential contribution of transcription to the response of dual DNA-PK/Top2 inhibition, immunohistochemical staining of transcription and replication status was assessed as a function of depth into the 3D spheroids and these results were compared along with Top2α and Top2β staining profiles. DNA replication and RNA synthesis patterns were visualized via 'click' chemistry staining with EdU and EU respectively 19,20 . Our results showed that both Top2α and EdU staining were restricted to the edge of spheroids (< 50 µm) while Top2β and EU staining were expressed more ubiquitously, extending into deeper layers of spheroids (Fig. 3C,D). DNA damage was also assessed using p53BP1 in spheroids treated with etoposide ± DNA-PK inhibition. When etoposide was exposed to spheroids alone, p53BP1 staining was only detected in the first 50 µm of the spheroids, consistent with greater DNA damage occurring in the proliferating fraction of spheroid cell populations. However, when 1 µM AZD7648 or M3814 was added, the p53BP1 was extended further into the spheroids (50-150 µm) where DNA synthesis was reduced or absent, but where RNA synthesis was still active (Fig. 3E).
DNA-PK inhibition is synergistic with Top2 poisoning in vivo: resulting in delayed tumor xenograft growth but also dose-limiting toxicity. We next tested Top2 poisons ± DNA-PK inhibition www.nature.com/scientificreports/ therapy in mice bearing FaDu or A549 xenografts. Figure 4 shows the anti-tumor effect of DNA-PK inhibition via AZD7648 with the Top2 poisons etoposide or pegylated liposomal-doxorubicin (PLD) in A549 xenografts. The AZD7648 treatment alone had no anti-tumor effect (Fig. 4E). The relatively low dose of etoposide (5 mg/ kg 3×/week for 3 weeks) had little antitumor effect compared to vehicle controls, and adding the lowest dose of AZD7648 (10 mg/kg 3×/week for 3 weeks) produced no sensitization. However, a significant delay in time to 3× tumor volume was observed at a higher dose of AZD7648 (30 mg/kg 3×/week for 3 weeks) with etoposide, extending to 28 days relative to 19 days for etoposide alone (Fig. 4E). Further increasing the AZD7648 dose to 100 mg/kg could not be completed due to severe normal tissue toxicity requiring an early endpoint termination (7 days after initial treatment). Weight loss of mice in the combination treatment groups are seen to follow a similar dose-dependent pattern to the growth inhibition effects, with all the groups experiencing rapid weight loss, and the highest combination group of 100 mg/kg AZD7648 group exceeding 20% in the first week (Fig. 4B,F). www.nature.com/scientificreports/ This is consistent with the sensitivity of normal tissue cells relative to cancer cells shown with monolayer cultures (Fig. 2C). A549 tumor-bearing animals treated with 6 mg/kg PLD once per week for 3 weeks as a single agent had a significant tumor growth delay relative to controls with time to tumor volume tripling extended from 19 to 48 days (Fig. 4E). The addition of 100 mg/kg AZD7648 to PLD further delayed the tumor growth to 72 days relative to 48 days for PLD as a single agent (Fig. 4E). Moderate normal tissue toxicity was seen for PLD alone as well as the combination group, as all mice had to be provided with dough diet supplements in order to not exceed 20% weight loss in the treatment groups; control groups gained weight with the dough diet (Fig. 4D,G). Similar results are also seen in FaDu xenografts (data not shown).
AZD7648 enhances DNA damage in FaDu xenograft tumors treated with etoposide in both cycling and non-cycling cell populations. We performed immunohistochemical staining and analysis of FaDu xenograft tumors treated with etoposide ± AZD7648 to examine the location of DNA damage and proliferating cells relative to tumor vasculature. Single agent treatment with a relatively high dose of 40 mg/kg etoposide significantly induced DNA damage in FaDu tumors at 6h. Staining for p53BP1 was detected primarily in regions where functional blood vessels (CD31) and proliferating cells (EdU) are located (representative images shown in Fig. 5A). The addition of 30 mg/kg AZD7648 extended the region of cells with positive DNA damage, as p53BP1 staining is seen to be more intense, and is now also detected in regions with fewer blood vessels.
Our results showed the EdU signal of control tumors peaking close to blood vessels (< 50 µm) and gradually decreasing as the distance increases (Fig. 5A). This is consistent with features of the solid tumor microenvironment where cells close to blood vessels are proliferative while those farther away become non-proliferative and quiescent [21][22][23] . Quantitative analysis was done to map the p53BP1 staining intensity as a function of distance to nearest CD31-stained blood vessels. Staining of p53BP1 in drug-treated tumors increases to a peak close to blood vessels in treated groups, before decreasing again farther away. However, while the etoposide alone group shows p53BP1 staining returning to control levels at distances far from vessels, addition of the DNA-PK inhibitor AZD7648 results in greater levels of persistent p53BP1-labeled DNA damage (Fig. 5B). Significant p53BP1 intensity increases were observed between etoposide single treatment and combination groups for regions 'close to blood vessels' and 'away from blood vessels' (p ≤ 0.0001 and p ≤ 0.005 respectively) (Fig. 5C). These findings suggest that adding DNA-PK inhibitors sensitizes non-proliferative tumor cells to etoposide treatment in vivo.
Overall, we have demonstrated that DNA-PK inhibitors sensitize cells to Top2 poison treatment in 3 preclinical models: cell culture, 3D spheroids and tumor-bearing mice. Relative to Top2 inhibition alone, immunohistochemical staining and flow cytometry analysis indicate enhanced DNA damage in non-proliferating and G 1 phase cancer and normal tissue cells in treatments when combining etoposide and DNA-PK inhibitors. Clonogenic survival assays and in vivo tumor growth delay studies confirm that the observed increase in DNA damage translates to greater cell kill and longer tumor growth delay in combination treatments compared to Top2 poison alone, however dose-limiting toxicities were also observed. Together, these data demonstrate that combining DNA-PK inhibitors with Top2 poisons is an effective treatment approach that is able to kill nonproliferating, quiescent tumor cells that are otherwise resistant to Top2 poison treatments to result in greater anti-cancer effects. However, the observed concomitant increase in toxicity to non-cancerous cells suggests a need for targeting the cell-killing effects to tumor tissues.  www.nature.com/scientificreports/

Discussion
We have shown that DNA-PK inhibitors such as AZD7648 enhance the anti-cancer effects of Top2 poisons by expanding the population of targeted cells to include the non-proliferating, but transcriptionally active cells prevalent in the tumor microenvironment. Top2 poison cytotoxicity is mediated by trapping Top2 proteins on the broken ends of DNA, with DNA DSBs generated as the protein-DNA complex collides with the machinery of replication or transcription. Evidence has shown that Top2α is the major isoform expressed in proliferating cells to guide replication, while Top2β is    www.nature.com/scientificreports/ expressed in all cell types to guide transcription 24,25 . Both isoforms can generate DNA DSBs when trapped by Top2 poisons, though Top2α mediated replication-dependent mechanisms seem to be the main cause of Top2 poison cytotoxicity 26,27 . Our DNA damage and cell survival results are consistent with these patterns. Cell culture and 3D spheroid models showed Top2α is mostly expressed in proliferating cells and treatment with etoposide increased DNA damage in this population, while the non-proliferating fraction is 10 times more resistant. Further, data from spheroids shows a dose-response relationship for etoposide cell-killing effect that plateaus with a significant > 40% surviving fraction even when the concentration is increased 10 times, suggesting a significant population of resistant cells (Figs. 1, 3). Top2β is the other Top2 isoform that is expressed in both proliferating and non-proliferating cells and mainly participates in transcription, as is confirmed in our spheroid data. The two isoforms share the same catalytic mechanism and are equally targeted by Top2 poisons 28,29 , however repair of the resulting DSBs is cell cyclespecific, with NHEJ involved in all phases of cell cycles and HR only activated during late S to G 2 phase. Our data again confirms what others have shown, that proliferating cells have greater sensitivity to Top2 poisons relative to non-proliferating cells with the implication that proliferating cells are less capable of repairing Top2 poisonrelated damage. Solid cancers contain a mixed population of proliferating and non-proliferating cells due to the complexity of the microenvironmental gradients of oxygen and nutrient supply, such that non-proliferating tumor cells distal to functional blood vessels are resistant to Top2α-targeted anti-cancer effects 30 . HR is a relatively slow but accurate process and a high dose of Top2 poison can induce DNA DSBs that can exceed capacity for HR repair 31 . The DSBs induced during transcription, on the other hand, may be more readily repaired via NHEJ, a repair process that is faster and has a larger capacity compared to HR 32 . Consequently, non-proliferative cells have greater opportunity to repair the DNA damage induced by Top2 poisons through NHEJ repair. Evidence from proteomic approaches further supports this hypothesis by showing that trapped Top2 is removed from DNA broken ends by different mechanisms during replication and transcription [33][34][35] . This results in different forms of DNA DSBs at 3′ end between replication and transcription (long vs short 3′ ssDNA overhangs) which favors HR and NHEJ repair respectively. Figure 6 summarizes the cellular responses to trapped Top2 for cells undergoing replication or transcription with their subsequent cell fates 36 .  www.nature.com/scientificreports/ Our data provides evidence to support the argument that NHEJ is the critical repair pathway for non-proliferating cells to repair the DNA DSBs introduced by Top2 poisoning treatment. Blocking the NHEJ pathway with the DNA-PK inhibitor AZD7648 sensitizes tumor cells to etoposide and doxorubicin, with the most significant impact on non-proliferating cells. Staining data in spheroids shows that Top2β is expressed ubiquitously in both proliferating and non-proliferating cells mapped via incorporation of EdU overlapping with the transcription profile of spheroids mapped using incorporation of EU. Addition of DNA-PK inhibitors enhanced the DNA damage of both proliferating and non-proliferative but transcriptionally active cells. This greater DNA damage translated to greater cell kill in vitro and increased anti-tumor effects in vivo, as demonstrated by the outcome of combining the DNA-PK inhibitor with Top2 poisons in clonogenic survival and tumor growth delay assays. Overall, our evidence suggests that Top2 poisons are most effective at killing cells unable to repair the damage, which appears to preferentially be the HR-dependent cells in S-phase. Quiescent cells that are able to use NHEJ to repair the DNA DSBs occurring during transcription by Top2β poisoning are less likely to die, however this can be overcome with inhibition of the key NHEJ protein, DNA-PK. Similarly, inhibiting other components of NHEJ pathway was also found to increase the cytotoxicity of Top2 poisons. Srivastava  150 µm Figure 6. Tumor derived quiescence leads to a population of Top2 poison resistant cells which can be targeted by DNA-PK inhibition. During replication, trapped Top2 is removed by replication run-off, leaving long single strand overhangs at the DNA 3′ end, which leads to classical Top2 poisoning-driven cellular death. During transcription, trapped Top2 is first degraded by 26S proteasome followed by Tdp2 cleavage, leaving DNA DSBs with two blunt ends that can be efficiently repaired by NHEJ. Inhibition of DNA-PK blocks the NHEJ pathway, leading to a novel mode of Top2-poison-derived cellular death. www.nature.com/scientificreports/ ligase IV inhibitor, SCR7, inhibits NHEJ pathway, and confers greater tumor control when treated in combination with etoposide. However, the detailed molecular mechanism resulting in the cytotoxicity and target cell populations was not investigated 37 . Despite the significant tumor growth delays achieved in our in vivo studies, the adverse effects in the combination treatment groups was evident. As most post-mitotic normal tissues are also non-proliferative but transcriptionally active, this combination therapy may result in a narrowing of the therapeutic index for Top2 poisons, limiting the range of doses where Top2 poisons can be effective without unacceptable adverse effects. Moderate to severe toxicities were observed in our combination treatment groups, where the highest dose of AZD7648 in combination with normally well tolerated 5 mg/kg etoposide induced excessive weight loss during the first week of treatment, requiring humane termination of animals in the study. The weight loss could be the result of excessive toxicity burden on the intestines, a highly proliferative organ with high sensitivity to toxic chemotherapy. Pegylated liposomal doxorubicin (PLD) has been found to have greater tumor-targeting specificity owing to a longer plasma half-life with lower peak concentrations 38 . Toxicity in the combination treatment groups still occurred with PLD, but was significantly less than that seen with etoposide. Other groups combining Top2 poisons with DNA-PK inhibitors have reported similar anti-cancer efficacy results, however they also report toxicity, with 15% 12 and 10% 11 body weight loss for animals treated with PLD (2.5 mg/kg) + AZD7648 (37.5 mg/ kg) relative to PLD alone. However, this toxicity can be overcome when a more localized chemotherapy is applied, such as using chemotherapy drug-loaded DC M1 polymer beads 39 . Other methods to limit toxicity and establish a therapeutic ratio in the clinic involve careful dosing and treatment scheduling design, optimizing cancer cell kill while minimizing dose-limiting toxicities. Mathematical models that incorporate cell cycle, DNA damage and repair kinetics have been used to predict in vivo xenograft response and generate tolerable treatment dosing schedules, as has been used in the phase I clinical trial design for ATR inhibitor AZD6738 in combination with radiotherapy 40 . This approach may provide a pathway to effective combination with Top2 poisons with DNA damage repair inhibitors as well.
In conclusion, addition of DNA-PK inhibitors to Top2 poisons introduces a synergistic effect with the greatest impact on non-proliferating cells, sensitizing these otherwise resistant cells to result in greater or more persistent DNA damage and significant decreases in tumor cell survival. However, sensitization of non-cancerous, normal tissues and the overall greater dose-limiting toxicities observed with combination treatments suggest a more targeted therapeutic approach is necessary to maximise the potential benefit of combining Top2 poisoning with DNA-PK inhibition.