Browsing by Subject "NAD(P)H Dehydrogenase (Quinone)"
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Item Improving the Efficacy and Expanding the Application of NQO1-Bioactivated Therapeutics(2015-06-09) Moore, Zachary Ray; Brekken, Rolf A.; Boothman, David A.; Scaglioni, Pier Paolo; Gao, JinmingNADPH:quinone oxidoreductase-1 (NQO1)-bioactivated drugs, such as ß-lapachone (ß-lap), are powerful therapeutics for tumor-specific therapy. They react with NQO1, which is highly overexpressed in most solid tumors, to cause a futile redox cycle that results in devastating oxidative DNA damage and energy depletion in the form of ATP, NAD(H) and NADP(H) loss specifically in tumor cells. However, ß-lap suffers from inherent limitations shared by quinone therapeutics, most notably methemoglobinemia at high doses caused by non-specific oxidation of hemoglobin. My goal was to increase the efficacy of ß-lap at lower, well-tolerated doses without increasing normal tissue toxicity. Targeting the NAD+ synthesis pathway by inhibiting NAMPT prevented cells from surviving the metabolic stress of NAD+ and ATP depletion induced by PARP1 hyperactivation secondary to ß-lap treatment. This resulted in synergistic cancer cell death at normally sublethal doses of both ß-lap and NAMPT inhibitors, occurring through the same NAD+-Keresis mechanism as with ß-lap alone. On the other hand, synergy with PARP inhibitors occurred due to an increased accumulation of DNA double strand breaks, which was a result of inhibited repair of ß-lap-induced single strand breaks. In contrast to synergy observed with NAMPT inhibition, PARP inhibitors combined with ß-lap caused canonical caspase-mediated apoptosis. In addition to providing new treatments for further preclinical and clinical development, these studies elucidated the importance of NAD+ and ATP depletion in cell death induced by ß-lap. Furthermore, these treatment strategies increase the tumor specificity and widen the use of both NAMPT and PARP inhibitors, since in combination with ß-lap they are effective against all NQO1-overexpressing tumor cells. As a study in expanding the application of NQO1-bioactivated therapeutics, I have also demonstrated NQO1 overexpression and ß-lap sensitivity in atypical teratoid rhaboid tumors (ATRTs), a rare but deadly pediatric malignancy that can be targeted with NQO1-bioactivated therapeutics.Item Modulating DNA Damage Responses for Improved Breast Cancer Therapy(2015-04-06) Ilcheva, Mariya Ilcheva; Amatruda, James F.; Burma, Sandeep; Boothman, David A.; Story, MichaelThe main focus of this work is to improve the efficacy of breast cancer therapy, either by utilizing novel agents that induce specific types of DNA damage resulting in metabolic changes, or by modulating factors that are involved in the repair of toxic DNA double-strand breaks (DSBs). The detoxifying enzyme, NQO1 (NAD(P)H:quinone oxidoreductase-1), is a promising therapeutic target due to its over-expression in many solid cancers, and very low presence in normal cells. Agents, such as β-lapachone and deoxynyboquinone (DNQ), which target NQO1 enzyme to induce programmed necrosis in solid tumors, have shown great promise. However, they have not been able to reveal the full potential of an NQO1-activated anticancer agent due to their low solubility, and more potent tumor-selective compounds are needed. Based on its structure and mode of action, isobutyl-DNQ (IB-DNQ) was recently added to the spectrum of NQO1 substrates. IB-DNQ increases NQO1 processing, enhancing both the potency and the selectivity of its anticancer properties, making it a highly efficient NQO1 substrate, and thus an outstanding anticancer agent. IB-DNQ is a promising antitumor agent whose mechanism of action has not been elucidated yet. We found that IB-DNQ killed breast cancer cells in an NQO1-dependent manner with greater potency than β-lapachone or DNQ. IB-DNQ treatment caused extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion leading to µ-calpain-mediated cell death (NAD+-Keresis). Next, we tested for synergy between IB-DNQ and the base excision repair (BER) inhibitor, methoxyamine (MeOX). Methoxyamine potentiated IB-DNQ cytotoxicity and allowed the use of very low doses of IB-DNQ, thereby potentially reducing any side-effects. Future studies in vivo will be geared toward proving the equivalent antitumor efficacy of IB-DNQ to β-lapachone and DNQ, but with much greater potency at lower doses. In addition, this study examined factors that modulate the response of BRCA1/2-deficient breast cancers to PARP1 inhibitors. Interestingly, a significant number of BRCA1-deficient breast cancers exhibit aberrantly reduced expression of the Mre11 protein, an important player in DNA damage detection and repair. We assessed the role of Mre11 in the response of BRCA1-deficient breast cancers to PARP1 inhibitors (PARP1i) and found that Mre11 depletion resulted in a significant increase in radial chromosomal structures after IR or PARP1i treatments. These aberrations were indicative of a diminished capacity to repair DSBs by homologous recombination (HR) and subsequent repair of lesions by error-prone pathways, such as NHEJ. Loss of Mre11 abrogated HR repair pathway in BRCA1-deficient cancers, as seen with reduced Rad51 foci, and increased the sensitivity of these tumors to PARP1 inhibitors, thus Mre11 status may be an important prognostic factor in the treatment of BRCA1-deficient breast cancers with PARP inhibitors. Another factor whose inhibition might hyper-sensitize breast cancers to PARP1 inhibitors is CDK1/2 due to the regulatory role that these kinases play in HR. Therefore, we explored the inhibition of CDK1/2 activity as a way to sensitize BRCA1-proficient cancers to PAPR1 inhibition. We found that CDK1/2 inhibition further abrogated DSB repair in BRCA1-deficient cancers, leading to heightened sensitivity to PARP1 inhibitors. These results indicate that inhibition of CDK1/2 could create a state of "BRCAness", which could expand the efficacy of PARP1 inhibitors not only for BRCA1/2-deficient cancers, but also to BRCA1-proficient ones.Item NQO1-Bioactivatable Drugs at the Interface of Cancer Metabolism and the DNA Damage Response(2015-05-07) Chakrabarti, Gaurab; Burma, Sandeep; Brekken, Rolf A.; DeBerardinis, Ralph J.; Boothman, David A.Increased levels of reactive oxygen species (ROS) have been observed in multiple cancer types, where they are crucial for tumor biology. Concomitantly, tumor cells also have enhanced expression of antioxidant pathway proteins to detoxify excess ROS. Thus, a challenge for anti-cancer therapeutics is to fine-tune this delicate balance from ROS protection, to ROS production while sparing normal tissue from toxicity. The phase II detoxification enzyme, NAD(P)H:quinone oxidoreductase-1, NQO1, is dramatically overexpressed in many solid tumor types, including pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). The Boothman laboratory has demonstrated that NQO1 bioactivates a unique class of quinones, such as ß-lapachone (ß-lap) and deoxyniboqunine (DNQ), through a futile redox cycle to generate massive levels of superoxide radical to induce extensive DNA oxidative base damage, single strand breaks and poly(ADP-ribose) polymerase 1 (PARP1)-driven depletion of intracellular NAD+. However, tumor cell NADPH and glutathione (GSH) biogenesis can attenuate the efficacy of this class of drugs by blunting the ROS formation produced from the futile redox cycle. Therefore, it is increasingly important to identify and target tumor specific antioxidant defenses to sensitize cancer cells, but not normal tissue, to NQO1 bioactivatable drugs. The data presented in the first half of this dissertation demonstrate that targeting glutamine dependent transamination reactions depletes antioxidant defenses in PDA and sensitizes tumors, but not normal tissue, to ß-lap-induced programmed necrosis in vitro and in vivo. Downstream of ROS formation, another mechanism by which tumors can attenuate ß-lap efficacy is through the repair of DNA lesions, specifically through base excision repair (BER). The latter half of this thesis focuses on inhibiting BER in combination with ß-lap as a mechanism to drive PARP1 hyperactivation and synergistic killing of NQO1-expressing PDA, but not associated normal tissue.