Combined inhibition of Ref-1 and STAT3 leads to synergistic tumour inhibition in multiple cancers using 3D and in vivo tumour co-culture models.

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.

Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices.

Here, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1-) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1- lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies.

Targeting Base Excision Repair in Cancer: NQO1-Bioactivatable Drugs Improve Tumor Selectivity and Reduce Treatment Toxicity Through Radiosensitization of Human Cancer.

Ionizing radiation (IR) creates lethal DNA damage that can effectively kill tumor cells. However, the high dose required for a therapeutic outcome also damages healthy tissue. Thus, a therapeutic strategy with predictive biomarkers to enhance the beneficial effects of IR allowing a dose reduction without losing efficacy is highly desirable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in the majority of recalcitrant solid tumors in comparison with normal tissue. Studies have shown that NQO1 can bioactivate certain quinone molecules (e.g., ortho-naphthoquinone and ß-lapachone) to induce a futile redox cycle leading to the formation of oxidative DNA damage, hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), and catastrophic depletion of NAD+ and ATP, which culminates in cellular lethality via NAD+-Keresis. However, NQO1-bioactivatable drugs induce methemoglobinemia and hemolytic anemia at high doses. To circumvent this, NQO1-bioactivatable agents have been shown to synergize with PARP1 inhibitors, pyrimidine radiosensitizers, and IR. This therapeutic strategy allows for a reduction in the dose of the combined agents to decrease unwanted side effects by increasing tumor selectivity. In this review, we discuss the mechanisms of radiosensitization between NQO1-bioactivatable drugs and IR with a focus on the involvement of base excision repair (BER). This combination therapeutic strategy presents a unique tumor-selective and minimally toxic approach for targeting solid tumors that overexpress NQO1.