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Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with APX2009 as red indicator dye generated from Caspase 3 activity is shown overlayed with brightfield image for each treatment. Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Confirmation of APE1/Ref-1 and STAT3 expression in 3d and monolayer conditions in RP-B-O1 and RP-B-O2 cells. Basal growth conditions as described in the methods for each cell line were used for monolayer and 3D conditions. No significant differences in expression relative to actin were observed in either condition. Representatuive blot is shown in [A], and quantified data as the ratio of each protein to actin from 3 determinations is shown in [B,C].

Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with DMSO control red dye by itself, Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with APX2009 as red indicator dye generated from Caspase 3. Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction–oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1–specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-κB and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies.Significance:This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.

Structures and NMR analysis of novel ref-1 inhibitors APX2009 and APX2014. A. APX3330; NMR and HPLC as previously published.15-18 B. APX2009; HPLC: HPLC were performed using an Alltech Alltima column C18 5u, 250 x 5.6 mm, flow 1 mL/min at 40 .C. Elution was with a mobile phase of 15:10:75 water:A1:methanol where A1 was made using 700 mL of water, 300 mL methanol and 3mL trimethylamine to which phosphoric acid was added to bring the pH to 3.4. HPLC analysis showed a purity of >99%. NMR: 300 MHz NMR -conforms to structure. NMR (CDCl3) Î' 8.15 (m, 2H), 7.75 (m,2H), 6.2 (s, 1H), 4.1 (s, 3H), 3.6 (br d, 4H), 2.2 (t, 2H), 1.4 (m, 4H), 1.25 (br d, 4H), 0.85(t, 3H). B. APX2014; HPLC: HPLC analysis was performed as for APX2009, but using a Zorbax SB Phenyl C18 5u, 250 x 4.6 mm column. Elution was with a mobile phase of 25:10:65 water:A1:methanol where A1 was made using 700 mL of water, 300 mL methanol and 3mL trimethylamine to which phosphoric acid was added to bring the pH to 3.4. Flow 1 mL/min at 40 .C. and showed a purity of >99%. NMR: 300 MHz NMR -conforms to structure. NMR (CDCl3) Î' 8.8 (br s,1H), 8.1 (m, 2H), 7.75 (m, 2H), 6.7 (s, 1H), 4.15 (s, 3H), 3.9 (s, 3H), 2.2 (m, 2H), 1.4 (m, 2H), 0.85 (t, 3H).

Confirmation of cell death induction by caspase activity assay and PARP cleavage. Representative images for caspase activity via red fluorescence [A] for the data calculated and shown in Figure 2, each with matching brightfield images. Representative examples for T24 and UC3 cells are shown form cells treated with the growth-inhibitor EC50 concentrations for each drug, in each cell line. PARP cleavage was used to confirm the induction of apoptotic mechanisms by western blotting for cleaved PARP1 [B], with representative cell experiments in which UC3 cells were treated with EC50 and EC90 concentrations of each drug.

Legend for supplementary videos.

APE1/Ref-1 localization in human tumors. Additional examples of APE1/Ref-1 immunofluorescence in human bladder cancer specimens at 10X and 40X. Lower power (10X) [A] demonstrates transition from nuclear localized APE1/Ref-1 in the epithelial compartment (Epi) to cytosolic staining in the invasive regions of a human tumor, in the muscularis (Musc) in a single image, with high power (40x) views showing the cellular localization [B,C]. Additional examples in high power are shown in [D], from 3 different specimens (1-3), exclusively nuclear staining in the epithelial compartment (Epi) and cytosolic staining in the invasive satellite lesions (INV).

Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction–oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1–specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-κB and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies. Significance: This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.

With a plethora of molecularly targeted agents under investigation in cancer, a clear need exists to understand which pathways can be targeted simultaneously with multiple agents to elicit a maximal killing effect on the tumour. Combination therapy provides the most promise in difficult to treat cancers such as pancreatic. Ref‐1 is a multifunctional protein with a role in redox signalling that activates transcription factors such as NF‐κB, AP‐1, HIF‐1α and STAT3. Formerly, we have demonstrated that dual targeting of Ref‐1 (redox factor‐1) and STAT3 is synergistic and decreases cell viability in pancreatic cancer cells. Data presented here extensively expands upon this work and provides further insights into the relationship of STAT3 and Ref‐1 in multiple cancer types. Using targeted small molecule inhibitors, Ref‐1 redox signalling was blocked along with STAT3 activation, and tumour growth evaluated in the presence and absence of the relevant tumour microenvironment. Our study utilized qPCR, cytotoxicity and in vivo analysis of tumour and cancer‐associated fibroblasts (CAF) response to determine the synergy of Ref‐1 and STAT3 inhibitors. Overall, pancreatic tumours grown in the presence of CAFs were sensitized to the combination of STAT3 and Ref‐1 inhibition in vivo. In vitro bladder and pancreatic cancer demonstrated the most synergistic responses. By disabling both of these important pathways, this combination therapy has the capacity to hinder crosstalk between the tumour and its microenvironment, leading to improved tumour response.