*2.1. KJ-28d Inhibits Growth of Human NSCLC Cells In Vitro and In Vivo*

Among the reported novel PARP-1 inhibitors, we examined KJ-28d, as shown in Figure 1A, based on the prior demonstrations of its significant inhibitory activity against PARP-1, as well as antitumor activity in *BRCA*-deficient ovarian cancer cells [21]. Specifically, we assessed whether KJ-28d can inhibit the growth of *BRCA*-proficient cancer cells. Human NSCLC cells were treated with KJ-28d and growth inhibition was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. KJ-28d significantly inhibited the growth of A549, H1299, H460, and H1650 human NSCLC cells with a determined IC<sup>50</sup> value, as shown in Figure 1B, and induced the sub-G1 phase (apoptotic cell) in A549 and H1299 cells, as shown in Supplementary Figure S1.

**Figure 1.** KJ-28d inhibits tumor growth of A549 and H1299 xenografts in nude mice. (**A**) The chemical structure of the KJ-28d compound. (**B**) A549, H1299, H1650, and H460 human non-small cell lung cancer (NSCLC) cells were treated with KJ-28d at the indicated concentrations for 5 days, and cell viabilities were determined by the MTT assay. Data are presented as means ± standard deviation (SD) from at least three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus DMSO-treated control. (**C,D**) A549 cells and H1299 cells were subcutaneously injected into the thigh of the right hind leg of BALB/c nu/nu mice (*n* = 3 per group, A549; *n* = 4 per group, H1299). Two weeks after tumor cell injection, KJ-28d (10 mg/kg) or DMSO (control) was intraperitoneally administered once every 2 or 3 days for seven times in total. (**C**) Longest (L) and shortest (W) tumor axes were measured, and tumor volume (mm<sup>3</sup> ) was calculated as L × W<sup>2</sup> /2. Data shown represent average tumor volume (\* *p* < 0.05, \*\* *p* < 0.01). Results are shown as means ± SD. (**D**) The body weights of A549 and H1299 xenograft mice were determined once a week during the experiments. Data are shown as means ± SD. **Figure 1.** KJ-28d inhibits tumor growth of A549 and H1299 xenografts in nude mice. (**A**) The chemical structure of the KJ-28d compound. (**B**) A549, H1299, H1650, and H460 human non-small cell lung cancer (NSCLC) cells were treated with KJ-28d at the indicated concentrations for 5 days, and cell viabilities were determined by the MTT assay. Data are presented as means ± standard deviation (SD) from at least three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus DMSO-treated control. (**C,D**) A549 cells and H1299 cells were subcutaneously injected into the thigh of the right hind leg of BALB/c nu/nu mice (*n* = 3 per group, A549; *n* = 4 per group, H1299). Two weeks after tumor cell injection, KJ-28d (10 mg/kg) or DMSO (control) was intraperitoneally administered once every 2 or 3 days for seven times in total. (**C**) Longest (L) and shortest (W) tumor axes were measured, and tumor volume (mm<sup>3</sup> ) was calculated as L <sup>×</sup> <sup>W</sup><sup>2</sup> /2. Data shown represent average tumor volume (\* *p* < 0.05, \*\* *p* < 0.01). Results are shown as means ± SD. (**D**) The body weights of A549 and H1299 xenograft mice were determined once a week during the experiments. Data are shown as means ± SD.

We next determined whether the antitumor effect associated with the in vitro KJ-28d treatment could be translated into a similar effect in an in vivo xenograft mouse model. BALB/c-nu/nu mice were subcutaneously (*s.c.*) implanted with A549 or H1299 cells in the right hind leg, and when tumors were palpable (average diameter approximately 150 mm<sup>3</sup> ; 10 days post-implantation), mice were intraperitoneally (*i.p.*) administered a dose of 10 mg/kg KJ-28d or DMSO (control vehicle) once every 2 or 3 days for a total of seven times. KJ-28d treatment inhibited A549 or H1299 cell-derived tumor growth by 51% and 49%, respectively, as compared with the respective vehicle, as shown in Figure 1C. Additionally, to determine the toxicity of KJ-28d, we measured the body weight of mice. Mice treated with KJ-28d did not show any difference in body weight as compared with control mice, as shown in Figure 1D. The results suggested that KJ-28d has antitumor activity for NSCLC cells in vitro and in vivo. We next determined whether the antitumor effect associated with the in vitro KJ-28d treatment could be translated into a similar effect in an in vivo xenograft mouse model. BALB/c-nu/nu mice were subcutaneously (*s.c.*) implanted with A549 or H1299 cells in the right hind leg, and when tumors were palpable (average diameter approximately 150 mm<sup>3</sup> ; 10 days post-implantation), mice were intraperitoneally (*i.p.*) administered a dose of 10 mg/kg KJ-28d or DMSO (control vehicle) once every 2 or 3 days for a total of seven times. KJ-28d treatment inhibited A549 or H1299 cell-derived tumor growth by 51% and 49%, respectively, as compared with the respective vehicle, as shown in Figure 1C. Additionally, to determine the toxicity of KJ-28d, we measured the body weight of mice. Mice treated with KJ-28d did not show any difference in body weight as compared with control mice, as shown in Figure 1D. The results suggested that KJ-28d has antitumor activity for NSCLC cells in vitro and in vivo.

#### *2.2. KJ-28d Induces DNA Damage and Generation of ROS in NSCLC Cells 2.2. KJ-28d Induces DNA Damage and Generation of ROS in NSCLC Cells*

Since PARPi induce accumulation of DNA damage [22,23], we sought to determine whether KJ-28d could induce DNA damage in NSCLC cells. The DNA damage was measured in A549 and H1299 cells at different time points after KJ-28d treatment by detecting the phosphorylation on Ser139 of the H2AX (γ-H2AX) histone protein, which is an indicator of the presence of DNA double-strand damage. KJ-28d induced γ-H2AX in both cell types at the latest time point (24 h), as Since PARPi induce accumulation of DNA damage [22,23], we sought to determine whether KJ-28d could induce DNA damage in NSCLC cells. The DNA damage was measured in A549 and H1299 cells at different time points after KJ-28d treatment by detecting the phosphorylation on Ser139 of the H2AX (γ-H2AX) histone protein, which is an indicator of the presence of DNA double-strand damage. KJ-28d induced γ-H2AX in both cell types at the latest time point (24 h), as shown in Figure 2A. As treatment with KJ-28d resulted in inducing a DNA damage response, we also investigated whether KJ-28d could augment ROS generation in NSCLC cells. Flow cytometry analysis showed that treatment of A549

and H1299 cells with 5 µM KJ-28d led to distinctly increased ROS levels, which were then reduced following treatment with *N*-acetyl-L-cysteine (NAC), a general free radical scavenger, as shown in Figure 2B,C. These results suggested that KJ-28d is able to exhibit antitumor activities in NSCLC cells through the accumulation of DNA damage and the generation of ROS. analysis showed that treatment of A549 and H1299 cells with 5 μM KJ-28d led to distinctly increased ROS levels, which were then reduced following treatment with *N*-acetyl-L-cysteine (NAC), a general free radical scavenger, as shown in Figure 2B,C. These results suggested that KJ-28d is able to exhibit antitumor activities in NSCLC cells through the accumulation of DNA damage and the generation of ROS.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 13

shown in Figure 2A. As treatment with KJ-28d resulted in inducing a DNA damage response, we also investigated whether KJ-28d could augment ROS generation in NSCLC cells. Flow cytometry

**Figure 2.** KJ-28d induces DNA damage and generation of reactive oxygen species (ROS). (**A**) A549 and H1299 cells were treated with 5 μM KJ-28d at indicated time points and immunoblotted for the detection of expression of γ-H2AX. (**B,C**) A549 and H1299 cells were treated with 5 mM NAC for 1 h, followed by KJ-28d for 24 h after incubation with 2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFHDA) for 30 min. Total cellular ROS production was measured using flow cytometry. Data are representative of three independent experiments (**B**). The bar graph shows the quantitative analysis of FACS data (**C**). Data are presented as the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01 versus corresponding values. **Figure 2.** KJ-28d induces DNA damage and generation of reactive oxygen species (ROS). (**A**) A549 and H1299 cells were treated with 5 µM KJ-28d at indicated time points and immunoblotted for the detection of expression of γ-H2AX. (**B,C**) A549 and H1299 cells were treated with 5 mM NAC for 1 h, followed by KJ-28d for 24 h after incubation with 20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFHDA) for 30 min. Total cellular ROS production was measured using flow cytometry. Data are representative of three independent experiments (**B**). The bar graph shows the quantitative analysis of flow cytometer data (**C**). Data are presented as the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01 versus corresponding values.

**Commented [AS3]:** Please define if appropriate.

#### *2.3. KJ-28d Potentiated Ionizing Radiation-Induced DNA Damage and Radiosensitized NSCLC Cells 2.3. KJ-28d Potentiated Ionizing Radiation-Induced DNA Damage and Radiosensitized NSCLC Cells*

As IR induces severe DNA damage, which can lead to overloading DNA repair capacity, it has been reported that PARP inhibitors enhance IR-induced DNA damage [14,17,20,22]. To examine whether KJ-28d could induce increased DNA damage in combination with IR, DNA damage was measured in A549 and H1299 cells treated with KJ-28d and IR by detecting the presence of γ-H2AX. Immunoblot analysis revealed a significant increase in the phosphorylation levels of H2AX protein as compared with KJ-28d or IR alone. Similarly, we observed high levels of staining of γ-H2AX foci in A549 and H1299 cells treated with both KJ-28d and IR, as shown in Figure 3A–C. PARylation by PARP-1 catalytic activity is a post-translational modification involved in DNA damage repair. To determine whether KJ-28d suppresses cellular PARylation, H1299 cells were treated with the indicated concentrations of either KJ-28d or olaparib, and A549 cells were treated with 5 μM KJ-28d with or without IR. We observed that 10 μM KJ-28d and 5 and 10 μM olaparib inhibited protein As IR induces severe DNA damage, which can lead to overloading DNA repair capacity, it has been reported that PARP inhibitors enhance IR-induced DNA damage [14,17,20,22]. To examine whether KJ-28d could induce increased DNA damage in combination with IR, DNA damage was measured in A549 and H1299 cells treated with KJ-28d and IR by detecting the presence of γ-H2AX. Immunoblot analysis revealed a significant increase in the phosphorylation levels of H2AX protein as compared with KJ-28d or IR alone. Similarly, we observed high levels of staining of γ-H2AX foci in A549 and H1299 cells treated with both KJ-28d and IR, as shown in Figure 3A–C. PARylation by PARP-1 catalytic activity is a post-translational modification involved in DNA damage repair. To determine whether KJ-28d suppresses cellular PARylation, H1299 cells were treated with the indicated concentrations of either KJ-28d or olaparib, and A549 cells were treated with 5 µM KJ-28d with or without IR. We observed that 10 µM KJ-28d and 5 and 10 µM olaparib inhibited protein PARylation in H1299, as shown in Figure 3D, and 5 µM of KJ-28d inhibited IR-induced PARylation in A549 cells, as shown in Figure 3E.

in A549 cells, as shown in Figure 3E.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 5 of 13

**Figure 3.** KJ-28d potentiates ionizing radiation (IR)-induced DNA damage responses. A549 and H1299 cells were treated with 5 μM KJ-28d 2 h before IR (4 Gy) and incubated for 24 h. The cell lysates were subjected to immunoblotting for detection of γ-H2AX (**A**), whereas cells were immunostained for γ-H2AX foci (red) and nuclei (DAPI: blue). Images were captured at 400× magnification. Scale bar: 20 μm (**B**). Quantification of the number of γ-H2AX foci per cell (**C**). Data represent the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus corresponding cells. (**D**) H1299 cells were treated with KJ-28d at indicated concentrations for 1 h. (**E**) A549 cells were treated with 5 μM KJ-28d and IR (4 Gy) and incubated for 1 h. The cell lysates were immunoblotted for the detection of expression of PAR. β-actin was used as a loading control. **Figure 3.** KJ-28d potentiates ionizing radiation (IR)-induced DNA damage responses. A549 and H1299 cells were treated with 5 µM KJ-28d 2 h before IR (4 Gy) and incubated for 24 h. The cell lysates were subjected to immunoblotting for detection of γ-H2AX (**A**), whereas cells were immunostained for γ-H2AX foci (red) and nuclei (DAPI: blue). Images were captured at 400× magnification. Scale bar: 20 µm (**B**). Quantification of the number of γ-H2AX foci per cell (**C**). Data represent the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus corresponding cells. (**D**) H1299 cells were treated with KJ-28d at indicated concentrations for 1 h. (**E**) A549 cells were treated with 5 µM KJ-28d and IR (4 Gy) and incubated for 1 h. The cell lysates were immunoblotted for the detection of expression of PAR. β-actin was used as a loading control.

Since KJ-28d potentiated IR-induced DNA damage in NSCLC cells, we further examined whether KJ-28d inhibited IR-induced cell growth. A549 and H1299 cells were treated with KJ-28d 2 h before IR. The clonogenic survival assay revealed that KJ-28d radiosensitized both cell lines, as shown in Figure 4A. Dose enhancement ratios (DER) of 0.75 μM KJ-28d-treated (at a surviving fraction of 0.37) to DMSO-treated cells were 1.5 and 1.23 in A549 and H1299 cells, respectively. We next determined whether treatment with KJ-28d could induce apoptotic cell death in A549 and H1299 cells. Apoptotic cell populations of these cell lines were detected using FACS analysis with annexin V/propidium iodide (PI) staining. Following treatment with 5 μM of KJ-28d and IR, the number of A549 and H1299 cells undergoing both early-stage (annexin V-positive/PI-negative) and late-stage (annexin V-positive/PI-positive) apoptosis increased significantly by 1.5-fold compared to KJ-28d alone, respectively, as shown in Figure 4B. In addition, KJ-28d plus IR treatment increased the cleavage of caspase-3 in both cell lines, as shown in Figure 4C. Taken together, these results indicated that KJ-28d enhanced both IR-induced DNA damage and apoptotic cell death in A549 and H1299 human NSCLC cells. Since KJ-28d potentiated IR-induced DNA damage in NSCLC cells, we further examined whether KJ-28d inhibited IR-induced cell growth. A549 and H1299 cells were treated with KJ-28d 2 h before IR. The clonogenic survival assay revealed that KJ-28d radiosensitized both cell lines, as shown in Figure 4A. Dose enhancement ratios (DER) of 0.75 µM KJ-28d-treated (at a surviving fraction of 0.37) to DMSO-treated cells were 1.5 and 1.23 in A549 and H1299 cells, respectively. We next determined whether treatment with KJ-28d could induce apoptotic cell death in A549 and H1299 cells. Apoptotic cell populations of these cell lines were detected using flow cytometer analysis with annexin V/propidium iodide (PI) staining. Following treatment with 5 µM of KJ-28d and IR, the number of A549 and H1299 cells undergoing both early-stage (annexin V-positive/PI-negative) and late-stage (annexin V-positive/PI-positive) apoptosis increased significantly by 1.5-fold compared to KJ-28d alone, respectively, as shown in Figure 4B. In addition, KJ-28d plus IR treatment increased the cleavage of caspase-3 in both cell lines, as shown in Figure 4C. Taken together, these results indicated that KJ-28d enhanced both IR-induced DNA damage and apoptotic cell death in A549 and H1299 human NSCLC cells.

**Commented [AS4]:** Please define if appropriate.

**Figure 4.** KJ-28d enhances the radiosensitivity of A549 and H1299 cells. (**A**) A549 and H1299 cells were treated with 0.75 μM KJ-28d for 2 h before IR (0, 1, 2, and 4 Gy). Clonogenic survival was measured 10 days after IR. Data are expressed as mean ± SD (*n* = 3) of the surviving fraction compared to non-irradiated cells. Colonies consisting of more than 50 cells were scored as survival colonies. (**B**) A549 and H1299 cells were treated with either 5 μM KJ-28d or 5 μM olaparib plus IR (4 Gy) for 48 h. Apoptotic cells were determined using the APC-conjugated annexin V/PI staining. Cell populations were gated into four groups, as described in Section 4. Bar graphs represent the mean percentage of early (annexin V-positive/PI-negative) and late apoptotic cells (annexin V-positive/PI-positive). Data represent the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01 versus corresponding cells. (**C**) A549 and H1299 cells were treated with 5 μM KJ-28d plus IR (4 Gy), and the cell lysates were subjected to immunoblotting for detection of cleaved caspase-3. β-actin was used as a loading control. **Figure 4.** KJ-28d enhances the radiosensitivity of A549 and H1299 cells. (**A**) A549 and H1299 cells were treated with 0.75 µM KJ-28d for 2 h before IR (0, 1, 2, and 4 Gy). Clonogenic survival was measured 10 days after IR. Data are expressed as mean ± SD (*n* = 3) of the surviving fraction compared to non-irradiated cells. Colonies consisting of more than 50 cells were scored as survival colonies. (**B**) A549 and H1299 cells were treated with either 5 µM KJ-28d or 5 µM olaparib plus IR (4 Gy) for 48 h. Apoptotic cells were determined using the APC-conjugated annexin V/PI staining. Cell populations were gated into four groups, as described in Section 4. Bar graphs represent the mean percentage of early (annexin V-positive/PI-negative) and late apoptotic cells (annexin V-positive/PI-positive). Data represent the mean ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus corresponding cells. (**C**) A549 and H1299 cells were treated with 5 µM KJ-28d plus IR (4 Gy), and the cell lysates were subjected to immunoblotting for detection of cleaved caspase-3. β-actin was used as a loading control.

#### *2.4. The Combination of KJ-28d and DNA Damage-Inducing Chemotherapeutic Agents Synergistically Inhibits NSCLC Cell Growth. 2.4. The Combination of KJ-28d and DNA Damage-Inducing Chemotherapeutic Agents Synergistically Inhibits NSCLC Cell Growth*

In preclinical and clinical studies of advanced NSCLC treatment, administration of PARPi in combination with DNA-damaging therapeutic agents, such as platinum-based compounds, taxane-based compounds, and topoisomerase inhibitors, has demonstrated enhanced cytotoxicity [24–26]. As KJ-28d enhanced radiosensitivity of A549 and H1299 cells, we expected that KJ-28d could increase cytotoxicity when combined with treatment with DNA DSB-inducing agents. A549 and H1299 cells were treated with KJ-28d and carboplatin, cisplatin, paclitaxel, or doxorubicin and evaluation of the synergistic effect of each pair-compound on cell viability was assessed using the MTT assay. Compared to treatment with KJ-28d or each compound alone, combination treatments showed a strong synergistic effect in both A549 and H1299 cells, decreasing cell viability in a dose-dependent manner, as shown in Figure 5A,C. To interpret the effects of all drug combinations, cell viability observed with each concentration of a KJ-28d-compound pair was converted to a combination index (CI) score using the CompuSyn software by Chou–Talalay [27]. CI scores were categorized as synergistic (CI < 0.9, green), additive (1.1 > CI ≥ 0.9, blue), or antagonistic (CI > 1.1, gray). We observed synergistic growth inhibition with a wide range of concentrations of KJ-28d and DSB-inducing agents in both cell lines, as shown in Figure 5B,D. Taken together, these results supported the conclusion that KJ-28d enhances the sensitivity of NSCLC cells to IR or chemotherapeutic agents of DNA-induced damage. In preclinical and clinical studies of advanced NSCLC treatment, administration of PARPi in combination with DNA-damaging therapeutic agents, such as platinum-based compounds, taxane-based compounds, and topoisomerase inhibitors, has demonstrated enhanced cytotoxicity [24–26]. As KJ-28d enhanced radiosensitivity of A549 and H1299 cells, we expected that KJ-28d could increase cytotoxicity when combined with treatment with DNA DSB-inducing agents. A549 and H1299 cells were treated with KJ-28d and carboplatin, cisplatin, paclitaxel, or doxorubicin and evaluation of the synergistic effect of each pair-compound on cell viability was assessed using the MTT assay. Compared to treatment with KJ-28d or each compound alone, combination treatments showed a strong synergistic effect in both A549 and H1299 cells, decreasing cell viability in a dose-dependent manner, as shown in Figure 5A,C. To interpret the effects of all drug combinations, cell viability observed with each concentration of a KJ-28d-compound pair was converted to a combination index (CI) score using the CompuSyn software by Chou–Talalay [27]. CI scores were categorized as synergistic (CI < 0.9, green), additive (1.1 > CI ≥ 0.9, blue), or antagonistic (CI > 1.1, gray). We observed synergistic growth inhibition with a wide range of concentrations of KJ-28d and DSB-inducing agents in both cell lines, as shown in Figure 5B,D. Taken together, these results supported the conclusion that KJ-28d enhances the sensitivity of NSCLC cells to IR or chemotherapeutic agents of DNA-induced damage.

**Figure 5.** The combination of KJ-28d and DNA damage-inducing chemotherapeutic agents synergistically inhibits growth of A549 and H1299 cells. A549 (**A**) and H1299 (**C**) cells were treated with the single or combined administration of KJ-28d and carboplatin, cisplatin, paclitaxel, or doxorubicin at indicated concentrations. Cell viability was determined 5 days after the treatment by MTT assay. Relative viability (normalized to DMSO-treated cells) is shown for each combination at indicated concentrations. Data are from one representative experiment of three independently repeated experiments. (**B,D**) Summary of tables showing combination index (CI) scores of KJ-28d and each chemotherapeutic drug combined at indicated concentrations in A549 (**B**) and H1299 cells (**D**). CI scores were calculated using the CompuSyn software and categorized as synergistic (CI < 0.9, green), additive (1.1 > CI ≥ 0.9, blue), or antagonistic (CI ≥ 1.1, gray). Each CI score was one representative datum from treatment with the indicated concentrations of single- and paired compounds from more than three independent experiments. **3. Discussion Figure 5.** The combination of KJ-28d and DNA damage-inducing chemotherapeutic agents synergistically inhibits growth of A549 and H1299 cells. A549 (**A**) and H1299 (**C**) cells were treated with the single or combined administration of KJ-28d and carboplatin, cisplatin, paclitaxel, or doxorubicin at indicated concentrations. Cell viability was determined 5 days after the treatment by MTT assay. Relative viability (normalized to DMSO-treated cells) is shown for each combination at indicated concentrations. Data are from one representative experiment of three independently repeated experiments. (**B,D**) Summary of tables showing combination index (CI) scores of KJ-28d and each chemotherapeutic drug combined at indicated concentrations in A549 (**B**) and H1299 cells (**D**). CI scores were calculated using the CompuSyn software and categorized as synergistic (CI < 0.9, green), additive (1.1 > CI ≥ 0.9, blue), or antagonistic (CI ≥ 1.1, gray). Each CI score was one representative datum from treatment with the indicated concentrations of single- and paired compounds from more than three independent experiments.

#### We previously identified KJ-28d as a novel PARP inhibitor that leads to increased cytotoxicity **3. Discussion**

in human ovarian cancer *BRCA-1*-deficient SNU-251 (*BRCA1* mutation at 5564G>A) cells, as well as in triple-negative human ovarian cancer *BRCA1* heterozygous (*BRCA1*+/−) MDA-MB-231 cells [21]. In this study, we further investigated the antitumor activity of KJ-28d on *BRCA*-proficient NSCLC cancer cells, as well as a combination approach with DNA damage-inducing agents to evaluate the synergistic therapeutic efficacy in human NSCLC cells. We previously identified KJ-28d as a novel PARP inhibitor that leads to increased cytotoxicity in human ovarian cancer *BRCA-1*-deficient SNU-251 (*BRCA1* mutation at 5564G>A) cells, as well as in triple-negative human ovarian cancer *BRCA1* heterozygous (*BRCA1*+/−) MDA-MB-231 cells [21]. In this study, we further investigated the antitumor activity of KJ-28d on *BRCA*-proficient NSCLC cancer cells, as well as a combination approach with DNA damage-inducing agents to evaluate the synergistic therapeutic efficacy in human NSCLC cells.

NSCLC is a type of cancer with a high mortality rate. Most patients with NSCLC receive treatment with platinum-based drugs, such as cisplatin or carboplatin, as first-line standard therapy. However, many patients who initially benefit from such chemotherapies gradually acquire chemoresistance. Thus, the development of novel therapies and strategies, including combination therapies, is needed. KJ-28d can inhibit the growth of various cancer cells, including NSCLC, breast, and colorectal cancer cells, as shown in Supplementary Figure S2. We specifically focused on NSCLC and revealed its antitumor effects on A549 and H1299 cells both in vitro and in vivo. A549 cells have a *KRAS* oncogenic mutation with c-Myc amplification, while H1299 cells are *TP53* mutant cells, as shown in Supplementary Table S1. Many studies have shown that KRAS-driven c-Myc amplification and TP53 correlate with genomic instability, thereby compromising DNA damage repair (DDR), causing the cells to be vulnerable to DDR inhibiting agents [28]. We observed that KJ-28d induced DNA damage and ROS generation in A549 and H1299 cells, which may facilitate cell death of NSCLC cells. The suppressed cellular PARylation might contribute to inducing DNA damage responses. Recent studies have suggested that PARPi can induce ROS generation due to DNA double stranded breaks. This is considered a characteristic of PARP inhibitors in DNA repair-deficient cells, as well as tumor suppressors or oncogene-mutated cells. Thus, PARPi may crosstalk with other signals [20,29]. In that regard, FDA-approved PARPi compounds have been studied to expand clinical use of PARPi in NSCLC [26,30,31].

Although the rationale for the use of PARPi was based on the reported induced synthetic lethality shown in *BRCA*-deficient cancer cells, preclinical and clinical studies suggested that PARPi could potentially be used as a combination partner with DNA-damaging agents in *BRCA*-proficient cancer cells [26,32,33]. Approximately 50% of patients with NSCLC receive radiotherapy during their treatment course [34]. Radiotherapy is an effective treatment modality, causing severe DNA damage. However, it can only treat tumors at defined doses because of the side effects to surrounding normal tissues. Although PARPi is not yet considered as a radiosensitizer in patients with ovarian cancer harboring *BRCA1*/*2* mutations in the clinic, the combination of radiotherapy with PARPi could provide promising synergistic therapeutic effects. For that reason, it has been studied in human NSCLC xenografts [35–37]. Olaparib is the first PARPi approved for the treatment of refractory ovarian cancer harboring *BRCA1* or *BRCA2* mutations. We have previously shown that treatment with KJ-28d induced more frequent apoptotic cell death in *BRCA* mutated ovarian cancer cells than olaparib did. It was noteworthy in this study that the combination of KJ-28d and IR also induced significantly more apoptotic cell deaths in NSCLC cells compared to combination treatment with olaparib and IR. Since we initially identified KJ-28d as a PARPi, further studies need to be conducted to elucidate other modes of action, as they may be cytotoxic. Platinum-based agents are widely used for a broad range of solid tumors, including NSCLC, and are the most commonly studied combination partners of PARPi [26,31,38]. Likewise, our results demonstrated that KJ-28d significantly enhanced the sensitivity of A549 and H1299 cells to carboplatin or cisplatin. In addition, we observed synergistic cell growth inhibitions of both cell populations when treated with a combination of KJ-28d with either paclitaxel or doxorubicin that constitute strong DNA-damaging therapeutic agents.

Besides the demonstrated inhibitory PARP activity of KJ-28d, the mechanisms responsible for KJ-28d-induced cytotoxicity in wild-type *BRCA* cancer cells have not been fully uncovered in this study. We suggest that KJ-28d might be involved in the antitumor activity exhibited in NSCLC cells with wild-type *BRCA* based on the experimentally exhibited markedly induced DNA DSBs and ROS generation at the latest examined time point (24 h). Structurally, KJ-28d contains the hydroxamic acid moiety found in histone deacetylase inhibitors (HDACi), including suberoylanilide hydroxamic acid. The hydroxamic acid moiety of HDACi acts as a chelator for zinc ions in the active site of histone deacetylases. To elucidate the possibility of HDAC inhibition, we examined the inhibitory activities of 1–11 HDAC isoforms using in vitro enzyme assays but observed little inhibitory activities of HDACs at KJ-28d concentrations under 5 µM, as shown in Supplementary Table S2. Further structure–activity relationship studies are required to understand the inhibitory activities of PARP-1/2 as a PARPi and to investigate the mechanisms associated with KJ-28d-induced growth inhibition and apoptotic cell death in NSCLC cells.

In summary, KJ-28d was cytotoxic to *BRCA*-proficient cancer cells, including NSCLC cells. Treatment with KJ-28d before IR led to increased DNA damage responses compared to treatment with KJ-28d or IR alone, resulting in the induction of apoptotic cell death. The combination of KJ-28d with carboplatin, cisplatin, paclitaxel, or doxorubicin considerably inhibited cell proliferation. In this

context, KJ-28d might act as an effective anti-cancer therapeutic agent against both *BRCA*-deficient and -proficient cancer cells and might have further potential as an adjuvant when used in combination with radiotherapy or DNA-damaging agents. However, further investigations into the mechanisms of action of KJ-28d in cancer cells are warranted.
