Abstract 5505: Potent redox-based inhibition of human MGMT for improving the efficacy of anticancer alkylating agents

There is an urgent need for the design and discovery of new and potent inhibitors for the DNA repair protein MGMT (O6-Methylguanine-DNA-methyltransferase) in glioma therapy. MGMT is highly expressed in brain tumors, and plays a primary role in conferring resistance to alkylating agents. The psuedosubstrates for MGMT such as the O6-benzylguanine have not been successful in the clinic due to prolonged inhibition of DNA repair in the bone marrow stem cells. Recently, we showed that the anti-alcoholism drug, disulfiram (DSF) inhibits MGMT activity in the same way as ALDH by conjugating with the active-site cysteine 145 (Carcinogenesis 35, 692, 2014). DSF, a symmetrical molecule, is metabolized and split in half to yield dithiocarbamate residues. Since the dithiocarbamates resulting from DSF decomposition are the ultimate reactive species that inactivate the aldehyde dehydrogenase and other signaling targets, we surmised that dithiocarbamate derivatives by themselves will be active as MGMT inhibitors and exert anticancer activities. Therefore, we tested various dithiocarbamates (pyrrolidine dithiocarbamate (PDTC); diethyldithiocarbamate (EDTC); dimethyldithiocarbamate (MDTC) and dibenzyldithiocarbamate (BDTC) on MGMT activity, protein levels, and other redox-sensitive proteins such as the NF-κB and GSTP1. The cytotoxicity of these dithiocarbamates against the MGMT-proficient SF-188 glioblastoma cells was comparable with that of DSF, with the MDTC being most effective and the benzyl derivative BDTC least potent. Western blot analysis in HT29 and SF-188 cells revealed a concentration-dependent degradation of MGMT by the dithiocarbamates, similar to DSF. All dithiocarbamates except the BDTC were superior to DSF in degrading MGMT protein. MDTC was the most potent followed by PDTC and EDTC in depleting the MGMT protein from tumor cells. Further, MDTC was also effective in reducing the cellular levels of NF-κB protein. We also established that MDTC binds to the active site cysteine145 in MGMT leading to its inactivation. Currently, we have developed the pegylated PLGA nanoparticles loaded with MDTC or zinc-chelated MDTC to target the glioblastoma and other cancers. The efficacy of these formulations in intracranial glioma models developed in nude mice is being evaluated. The lack of cytotoxicity and inability to bind with MGMT by BDTC suggests that the bulky group attached to the dithiocarbamate may hinder interaction with target proteins or interfere with their metabolism. Since MDTC has a good potential to cross the blood brain-barrier, possess reactive thiol groups that can interact with not only MGMT but also numerous signaling proteins, our strategy using this repurposed compound holds promise in glioma treatment (supported by grants from CPRIT [RP130266] and Carson-Leslie Foundation to KSS). Citation Format: Hanumantha Rao Madala. Potent functional inactivation of the MGMT DNA repair protein by dithiocarbamate compounds increases the efficacy of temozolomide in human glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3722.

Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC50 between 20-75 nM) while the response to DS alone showed significant variation (IC50 from 20 nM - 7 µM). Cu2+ alone was not toxic. Cytarabine (Ara-C), a key drug in AML treatment, was compared with DS/Cu2+. The AML cell lines THP1 (acute monocytic leukemia), UT7-epo and CMY (acute megakaryoblastic leukemia) were profoundly resistant to Ara-C (IC50 between 1.2-2.4 µM) but showed extreme sensitivity to DS/Cu2+ (IC50 between 31-46 nM). Although DS is an inhibitor of ALDH, we demonstrated that the cytotoxicity of DS or DS/Cu2+ was not directly associated with the level of ALDH or its inhibition. AML cell lines were treated with different doses of DS or DS/Cu2+ and ALDH activity and cell death were determined by flow cytometry. The percentage of cell death was dose-dependent and occurred irrespective of ALDH activity in cell lines with high endogenous ALDH activity (MV-4-11, CMY, CMK and K562) compared to those with low or undetectable ALDH activity (Kasumi-1, Molm-13, NB4 and KG-1a). As DS-mediated cytotoxicity has been associated in previous reports with proteasome inhibition, the effects of DS and DS/Cu2+ were compared to Bortezumib. Western blot analysis showed that DS induced PARP cleavage similar to Bortezumib, suggesting that proteasome inhibition may be contributing to DS or DS/Cu2+ cytotoxicity. Conclusion These data demonstrate that multiple AML cell lines are sensitive to DS and to DS/Cu2+, including some that show extreme resistance to Ara-C, such as CMY from a patient with Down Syndrome. This DS sensitivity may provide a less toxic and efficacious treatment approach to be tested in this group of patients with poor outcome following relapse. The cytotoxic activity of DS does not appear to be solely dependent on ALDH inhibition. Ongoing studies are focusing on determining the mechanisms of cell death, and cytotoxic efficacy in in vitro and xenograft models using patient AML samples, with the goal of developing a clinical trial in patients with relapsed/refractory AML. Citation Format: Eiman A. Aleem, David O. Azorsa, Ranjan Bista, Oliver B. Pepper, David W. Lee, Daniel H. Wai, Robert J. Arceci. Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2014-3982

Atypical teratoid/rhabdoid tumors (AT/RT) are one of the most malignant pediatric brain tumors with a dismal prognosis. Cells with high aldehyde dehydrogenase (ALDH) activity from brain tumors have a number of characteristics that are expected of brain tumor initiating cells (BTICs). This study aimed to evaluate the therapeutic potential of ALDH inhibition using disulfiram (DSF) against BTICs from AT/RT. Primary cultured BTICs from AT/RT were stained with Aldefluor and isolated by fluorescence activated cell sorting. Therapeutic effect of DSF against BTICs from AT/RT was confirmed using in vitro and in vivo studies. AT/RT showed high expression of ALDH. DSF significantly inhibited ALDH enzyme activity of AT/RT cells. DSF decreased self-renewal ability, cell viability, proliferation potential and induced apoptosis and cell cycle arrest on ALDH+ AT/RT cells. DSF showed more potent cytotoxic effect to ALDH+ AT/RT cells compared to standard anti-cancer agents including ifosfamide, carboplatin and etoposide. Importantly, DSF reduced metabolite (metabolism?) of ALDH+ AT/RT cells by increasing NAD+ ratio and regulating SIRT1, NF-κB, Lin28A/B and miRNA let-7g. Notably, treatment with DSF did not have considerable effect on the normal neural stem cells and fibroblasts. Animals in the DSF-treated group demonstrated a significant survival benefit (105 days of median survival in the DSF-treated group versus 91 days in the control group, p = 0.0219). Our study demonstrated the therapeutic potential of DSF against BTICs from AT/RT and suggested the possibility of ALDH inhibition for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Seung Ah Choi, Jung Won Choi, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Yong Hwy Kim, Young-Hoon Kim, Sung-Hye Park, Seung-Ki Kim. Targeting brain tumor initiating cells in atypical teratoid/rhabdoid tumors: Aldehyde dehydrogenase inhibition with disulfiram. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2014-5513

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with limited treatment options. Currently two immunotherapies are approved for the treatment of MCC, the PD-L1 inhibitor avelumab and the PD-1 inhibitor pembrolizumab. Unfortunately, these treatments only have durable benefit for less than half of patients that can be treated with immunotherapy. Therefore, new and effective treatments are needed for MCC. To address this, we conducted a high-throughput drug screening of ~4,000 small molecules. Our drug screen identified disulfiram (DSF), an aldehyde dehydrogenase inhibitor used in the treatment of alcoholism, as an agent that selectively reduced MCC cell viability. Recent studies suggest that DSF complexed with copper can have anti-cancer effects. Consistent with this, the addition copper at physiological levels increased the potency of DSF in MCC cells lines. Next, we demonstrated that disulfiram combined with copper synergized with the topoisomerase II inhibitor etoposide, lowering the effective dose of etoposide required to reduce MCC cell viability. Mechanistically, combining DSF, copper and etoposide was cytotoxic but did not induce apoptosis in MCC cell lines. This combination increased the formation of double stranded DNA breaks while disulfiram plus copper induced autophagy, which could mechanistically be an indication of immunogenic cell death signaling. Taken together, our data suggest that disulfiram combined with copper and etoposide could be repurposed for the effective treatment of advanced MCC. One strong advantage of this combination is that all agents can be given orally and at dosages with minimal adverse effects. Citation Format: Natasha Tremayne Hill, Tara Gelb, Dan Urban, Tyler Kellenberger, Matthew Hall, Isaac Brownell. Disulfiram plus copper synergizes with etoposide for the treatment of Merkel cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 575.

The DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) is a major determinant of tumor resistance to alkylating agents. Current clinical strategies involving O6-benzylguanine (BG) to inhibit MGMT and increase the drug efficacy have not been successful due to cumulative and unmanageable bone marrow toxicity. Although the gene therapy of hematopoietic stem cells with BG-resistant MGMT has been proposed, such practices are likely expensive and unpractical for routine clinical use. Therefore, there is an urgent need for finding new and transient inhibitors of MGMT. In a new approach, we exploited the highly reactive nature of Cysteine 145 which accepts the alkyl groups for novel drug design. Cys145 has a pKa of 4.8 and is susceptible for thiolation and nitrosylation, both of which inactivate the MGMT. In line with this, nitroaspirin (NCX-4016, NA), a cardioprotective drug was found to be an extremely potent inhibitor of MGMT, far superior to BG. Nitroaspirin is degraded by plasma and tissue esterases to release NO and aspirin in tissues. NA at pharmacologically achievable concentrations of 10-20 μM caused 100% inhibition of MGMT within 1 h in a large panel of human cancer cell lines including gliomas. The nitrosylated MGMT protein disappeared completely within 2 h. These data are highly comparable and superior to those elicited by BG. Using mutant MGMT proteins, we showed the active site cysteine (Cys145) and a neighboring Tyr114, a residue critical for DNA repair, were both modified by nitroaspirin. The ubiquitin-proteasome pathway mediated the degradation of nitrosylated MGMT. In cultured cells, MGMT suppression by NA was less prolonged than BG (80% regeneration at 40 h post NA exposure compared to 20% that by BG in different cell lines). However, pre-exposure of tumor cells to NA followed by BCNU resulted in (i) 2.5-fold greater levels of DNA interstrand crosslinks, (ii) a 4-fold increased G2/M cell cycle arrest, and (iii) 2-4 fold increased cell killing in various tumor cell lines. A single injection of NA (100 mg/kg) caused 50-60% inhibition of MGMT activity in mouse brain and liver. Because NA is a very nontoxic (IC50 >500 μM for most cell lines), is lipophilic enough to cross the BBB, and its pleiotropic actions actually benefit chemotherapy, the creation of an MGMT-deficient state by NA holds unequivocal promise for improving the therapy of primary and metastatic brain tumors (supported by RO3 CA125872 and the Association for Research of Childhood Cancer). Citation Format: Kalkunte S. Srivenugopal, Ameya S. Paranjpe. The NO-releasing aspirin inactivates and degrades human MGMT more efficiently than O6-benzylguanine and greatly sensitizes brain tumor cells to alkylating agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4473. doi:10.1158/1538-7445.AM2013-4473

Introduction: Glioblastoma Multiforme (GBM) is the most common lethal brain tumor associated with dismal survival rate. It is widely accepted that GBM contains a small population of cancer cells (∼1%) expressing CSC markers that promotes therapeutic resistance. High levels of aldehyde dehydrogenase (ALDH) activity, a characteristic feature of CSCs, play a significant role in chemoresistance. Many recent studies have elucidated that intra-tumoral hypoxia induces CSC phenotypes in tumour via epithelial-to-mesenchymal transition (EMT). Hypoxia inducible factors (HIFs) are master transcriptional regulators under hypoxic conditions. However NF-κB, another key transcription factor, is also highly up-regulated in hypoxia induced CSCs. There are no clear insights on how HIFs and NF-κB together orchestrates the anti-apoptotic signalling, chemo-radiation resistance and maintenance of stemness in CSC phenotypes. Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) effectively reverses chemoresistance in many cancers. DS/Cu is a strong inhibitor of NF-κB and ALDH and induces apoptosis. In this study we used an in vitro GBM CSC model and NF-κB-p65 and HIFs transfected GBM cell lines to investigate the relationship between hypoxia induced HIFs, NF-κB activation and ALDH activity and their role in chemoresistance. We also examined the cytotoxicity of DS/Cu on GBM CSCs and its effect on the above pathways. Results In comparison with adhered cells, the GBM cellsgrown as spheres in specialized neurosphere medium (NS) or normal DMEM (SUS) showed high proportion of hypoxic cells and elevated levels of CSC and EMT markers (ALDH, CD133, CD44, OCT4, SOX2, NANOG, E-cadherin to N-cadherin switch) suggesting hypoxia induced EMT phenotypes inCSCs. Increased levels of HIFs, ALDH (1A3 and 3A1) and NF-κB activity were detected in CSCs. CSCs are highly resistant to conventional anticancer drugs like temozolomide, paclitaxel, vincristine and doxorubicin. GBM cells transfected with NF-κB-p65 displayed CSC markers, chemoresistance and nuclear translocation of HIFs. Although transfection of HIFs induced expression of CSC markers, the cells did not show NF-κB activation and are not resistant to anticancer drugs. These results indicate the pivotal role of NF-κB in maintaining CSC characteristics and chemoresistance. DS is cytotoxic to both GBM cell lines and the CSCs derived from them in a Cu-dependent manner.DS/Cu inhibits NF-κB and ALDH activity and triggers intrinsic apoptotic pathway. After short exposure (2 hours) to low concentration of DS/Cu, the CSCs are completely eradicated in the cell culture. Conclusions: NF-κB plays pivotal role in chemoresistance. DS/Cu inhibits NF-κB, eradicates CSCs and reverses resistance in GBM.DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the BBB. Further study may lead to quick translation of DS into clinical trials. Citation Format: Vinodh Kannappan, Peng Liu, Sarah Brown, Xiuwu Bian, Tawari Patricia Erebi, Angel L. Armesilla, John L. Darling, Weiguang Wang. Disulfiram targets glioblsatoma stem like cells by modulating aldehyde dehydrogenase and hypoxia-NF-κB pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 222. doi:10.1158/1538-7445.AM2014-222

Many anticancer drugs induce reactive oxygen species (ROS) in cancer cells. High ROS will activate JNK pathway and induce cancer cell apoptosis. Meanwhile, ROS also strongly induces NFκB activity which triggers expression of many antiapoptotic factors and inhibits ROS/JNK pathway therefore antagonises anticancer drug-induced programmed cell death. The fate of anticancer agent-treated cancer cells is highly determined by the cross-talk between NFκB and JNK pathways. In this study, we demonstrated that a clinically used anti-alcoholism drug, disulfiram (DS), simultaneously activated ROS-JNK pathway and inhibited NFκB. DS was cytotoxic to breast cancer cell lines in vitro. As a divalent metal ion chelator, the cytotoxic effect of DS on breast cancer cell lines was highly copper-dependent. In the medium containing physiological concentration of copper (Cu, 1µM) the IC50 concentrations of DS to breast cancer cell lines were 200 - 500nM. The chemosensitizing effect of DS on 3 anti-breast-cancer agents, gemecitabine (dFdC), doxorubicine (DOX) and paclitaxel (PAC), was determined in 3 breast cancer cell lines (MCF-7, MDA-MB-231 and T47D). In combination with DS/Cu, the cytotoxicity of dFdC, DOX and PAC was significantly enhanced (Table 1. DOX: 8 - 11-fold; dFdC: 1.2 - 4-fold; PAC: 4 - 10-fold). DS also reversed dFdC resistance in acquired dFdC resistant cell lines. CI-isobologram analysis demonstrated synergistic effect between DS and anticancer drugs. Flow cytometric analysis showed DS enhanced anticancer drug-induced apoptosis. DS enhanced anticancer drug-induced ROS activity. The phosphorylated c-Jun and JNK protein in breast cancer cell lines was significantly induced by exposure of cancer cells to DS/Cu complex. Transfection of NFκB p50 and p65 induced dFdC resistance in MCF7 cells. DS inhibited drug-induced IκBα degradation and NFκB activation. Our data suggested that DS may be developed as a chemosensitizer for BC chemotherapy. *Equal contributionTable 1.DS/Cu sensitised cytotoxicity of anticancer drugs to breast cancer cell linesTreatmentsDrug: DS/CuMCF7MDA-MB-231T47DDOX (nM) 440.5 (16.2)178.5 (11.9)160.0 (5.5)DOX + DS/Cu1:540.3 (9.3)22.0 (2.0)21.5 (3.1)dFdC (nM) 22.1 (3.4)12.3 (2.2)35.0 (4.3)dFdC + DS/Cu1:104.9 (0.5)11.3 (1.2)12.4 (0.7)PAC (nM) 4.3 (1.4)9.3 (0.7)2.6 (0.3)PAC + DS/Cu1:62.50.4 (0.1)0.6 (0.02)0.7 (0.1) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5410.

Background Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that the cancer cells expressing CSC markers are highly resistant to chemo-radiation therapy. Up to 60% of locally advanced solid tumours exhibit hypoxia. Accumulated evidence demonstrates that hypoxia induces epithelial-to-mesenchymal transition and CSCs phenotypes in cancer cells. The pathways between hypoxia and CSCs are still largely in the dark. NFκB is a transcription factor being involved in chemo-radiation resistance. Activation of NFκB by hypoxia in a wide range of cells in inflammatory conditions has been identified for more than a decade. Recently, emerging evidence demonstrates that NFκB is also a key factor maintaining the stemness in breast CSCs. Therefore NFκB may mediate and be involved in hypoxia-induced CSCs phenotypes. Our previous studies show that disulfiram (DS), an anti-alcoholism drug, reverses chemoresistance of a variety of anticancer drugs in several types of cancer. In this study, we used an in vitro model to investigate the relationship between hypoxia-NFκB pathway and CSC phenotypes in two breast cancer cell lines (T47D and MCF7). The effect of DS on hypoxia-NFκB pathway and the cytotoxicity of conventional anticancer drugs (paclitaxel, gemcitabine and doxorubicin) in CSCs were also observed. RESULTS A huge proportion of hypoxic cells were detected in mammospheres. NFκB activation (AKT phosphorylation, IκBα degradation, p65 nuclear translocation, phosphorylation and NFκB transactivation) was detected in the CSCs. The percentage of breast cancer cells with CSC markers (ALDH+, CD24low/CD44high) is significantly increased in the NFκB p65 transfected cell lines. The transfected cell lines are highly resistant to doxorubicin, paclitaxel and gemcitabine. In contrast, breast CSCs do not show any resistance to DS. The cytotoxicity of DS is copper-dependent and cancer specific with no cytotoxicity in normal cell lines. CI-isobologram analysis demonstrates that DS significantly enhances cytotoxicity of conventional anticancer drugs (doxorubicin: 8 - 11-fold; gemcitabine: 1.2 - 23.5-fold; paclitaxel: 4 - 10-fold). DS simultaneously activates ROS-JNK pathway and blocks p65 nuclear translocation. The effect of DS on stemness of breast cancer cell lines is confirmed by its elimination of ALDH+VE CD24Low/CD44High population in mammospheres. To prolong the half-life of DS in bloodstream, we recently developed a liposome encapsulated DS. The liposomal DS shows very strong anticancer activity in an animal cancer model. CONCLUSIONS Our results demonstrate that hypoxia-NFκB pathway is involved in maintaining the stemness and chemoresistance in breast CSCs. DS is highly cytotoxic to breast CSCs and enhances cytotoxicity of conventional anticancer drugs. This may be caused by simultaneous induction of ROS and inhibition of NFkB pathway. Citation Format: Peng Liu, Sarah Brown, Vinodh Kannappan, Angel L. Armesilla, John L. Darling, Weiguang Wang. Manipulation of hypoxia-NFκB pathway to target breast cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 227. doi:10.1158/1538-7445.AM2013-227 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies (as high as 80%), depending on the type and stage of the tumors (www-p53.iarc.fr). The majority of mutations are missense that are localized to six “hotspot” residues, which play a role either in the structural integrity (structural mutants, e.g., R175H) or in the DNA binding domain (contact mutants, e.g., R273H). Previously, we showed that phenethyl isothiocyanate (PEITC), a naturally occurring dietary constituent present abundantly in watercress, selectively depletes p53 mutant protein, but not the WT p53, and induces apoptosis in tumor cells expressing p53 mutants. In this study we demonstrated that PEITC can reactivate p53R175 mutant protein and inhibit tumor growth in a mutant p53R175H -dependent manner. PEITC displayed a 175 allele preference of inhibition of cell proliferation as compared to other hotspot mutants as detected by WST-1 assay and induced apoptosis in a p53R175 mutant -dependent manner. Immunofluorescence and co-immunoprecipitation assay on PEITC treated p53R175 cells using conformation-specific antibodies (PAB1620 and PAB240) showed restoration of the “WT-like” conformation to p53 mutant. Reactivation of the WT like functions to the p53R175 mutant was established by its ability to bind DNA, transactivate p53 regulated downstream target genes (p21, PUMA, MDM2, NOXA, BAX and BCL2), and overexpression of p21 protein. PEITC treatment induced DNA damage response as detected by pATM-S1981 and pCHK2-Thr68, G2/M and S-phase cell-cycle arrest and oxidative stress as measured by decrease in the GSH levels in the cells. In contrast, co-treatment of cells with PEITC and reducing agents alleviated the inhibitory effects of PEITC on cell proliferation. Elevated oxidative stress due to the combined effect of PEITC and elevated ROS levels in p53 mutant cells might be responsible for the activation of restored “WT-like” p53R175 mutant protein and induction of apoptosis. PEITC induced proteasomal degradation and autophagy of the p53R175 mutant. Animals fed with PEITC diet showed a statistically significant reduction in xenograft tumor volumes, proliferation marker Ki67, and mutant p53 stained cells. Elevated mRNA levels of p53 regulated genes from animals fed PEITC diet provide in vivo evidence for the p53R175H mutant reactivation and inhibition of xenograft growth in a mutant p53-dependent manner. These are important findings because they demonstrated that a dietary compound can inhibit the growth of tumor cells in vivo by restoring the tumor suppressor functions to mutant p53, and thus provide a target for the development of novel chemopreventive strategies through dietary interventions. Citation Format: Monika Aggarwal, Elizabeth Sinclair, Anna Jacobs, Ying Fu, Marcin Dyba, Xiantao Wang, Idalia M. Cruz, Deborah Berry, Kallakury Bhaskar, Susette C. Mueller, Maria Laura Avantaggiati, Fung-Lung Chung. Reactivation of p53R175 mutant by dietary phenethyl isothiocyanate (PEITC) impairs tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2015-1910

The blood and brain barrier (BBB) and chemoresistance are the major obstacles for the success of glioblastoma multiforme (GBM) chemotherapy. It has been shown that high nuclear factor kappa B (NFκB) activity is highly related to chemoresistance. Gemcitabine (dFdC) can easily cross the BBB and gets into GBM tissues. Due to the chemoresistance of GBM cells to dFdC induced cytotoxicity, this drug is currently only used as a radiosensitiser for GBM treatment. Disulfiram (DS) is an anti-alcoholism medicine. Our previous study demonstrates that DS strongly inhibits NFκB activity and sensitises cancer cell lines to several anticancer drugs including dFdC. DS can freely pass through BBB. Therefore, we tested the chemosensitising effect of DS on dFdC in three GBM cell lines (U251MG, U87MG and U373MG). DS was cytotoxic to GBM cell lines. Copper (Cu) was essential for DS induced cytotoxicity in GBM cell lines. Cu or DS alone was not cytotoxic to GBM cell lines in vitro. Whereas high cytotoxicity (IC50 = 150 - 250 nM) was demonstrated when DS was dosed in combination with physiological concentration of Cu (1 µM). We also tested the chemosensitising effect of DS/Cu complex on dFdC in GBM cell lines. The GBM cell lines were highly resistant to dFdC induced cell death. DS significantly sensitised the cytotoxicity of dFdC in GBM cell lines (∼ 6 - 23-fold; Table 1). CI-isobologram analysis indicated that the combination effect between DS/Cu and dFdC was highly synergistic (Table 1). Flow cytometric data demonstrated that DS/Cu enhanced dFdC induced apoptosis. The molecular chemosensitising mechanisms of DS/Cu to dFdC in GBM cells are still not full elucidated. Our preliminary data indicated that DS/Cu simultaneously inhibited NFκB pathway and triggered reactive oxygen species (ROS)-JNK pathway.Cytotoxicity of dFdC alone or dFdC plus DS/Cu in GBM cell lines U251MGU87MGU373MGdFdC alone100.4 (22.3)>40078.6 (5.6)dFdC + DS/Cu (1:10 ratio)17.5 (5.1)17.1 (3.0)6.8 (1.8)CI0.390.470.82 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5380.

Introduction: Mutations in TP53 are common in early pancreatic and ovarian cancer development, and are detected in the majority of pancreatic cancers (75%) and serous ovarian cancers (97%). Mutations in TP53 induce autoantibody (AAb) immune responses, which may be biomarkers for the early detection of these cancers. The most common mutations in p53 occur in the DNA binding domain at residues that directly contact the DNA (Arg175, 248, and 273). In contrast, preliminary studies suggest that mutations in the N- and C- termini of p53 alter the thermodynamic stability of p53, leading to increased accumulation in tumors. It is not known if the location of the mutations impacts immunogenicity, or if p53-AAb are specific to mutant p53 proteins. Methods: To evaluate AAb to mutant p53 proteins, we generated custom programmable protein microarrays displaying 52 common p53 point mutations. cDNAs encoding full-length p53 with c-terminal GST tags were printed on arrays with anti-GST antibody, and transcribed and translated in situ using HeLa expression lysate. The microarrays were screened with sera from pancreatic cancer cases (Stage I-III, n=60), healthy and benign disease controls (n=63), ovarian cancer cases (n=19) and controls (n=17). Bound IgG was detected by fluorescence. For validation, four immunogenic mutant p53 proteins Arg248Met, Pro278Ser, Arg213Leu, Gly187Ser and wild-type p53 were individually displayed in 96-well format using a programmable ELISA. The ELISAs were re-screened with a subset of the sera from pancreatic cancer cases (n=49) and controls (n=47) and all of the ovarian cancer cases and controls. Cutoff values were established to determine if a patient is positive for all wild-type and mutant p53 AAb by measuring the mean antigen florescence intensity +2 standard deviations of the controls. Results: AAb were detected to all four mutant and wild-type p53 proteins in sera from 8/49 (16%) of pancreatic cases and 2/47 (4.2%) of pancreatic controls, but the results were not statistically significant. In comparison, AAb to all four mutant and wild-type p53 proteins were identified in sera from 8/19 (42%) of ovarian cases and 0/17 (0%) of healthy control women (p ≤ 0.005). All sera with AAb to mutant p53 also had measurable AAb to wild-type p53 protein. All patients with immune responses had evidence of broad-reactivity to at least at least two mutant p53 proteins, as well as to wild-type p53. Conclusion: Our results indicate that patients with serum AAb to wild type p53 protein also have AAb to multiple mutant p53 antigens, suggesting degeneracy of the epitope specificity. No patients have been identified with AAb specific to mutant-only p53. We observed inter-patient variation in the patterns of immune reactivity to p53 mutations that is of unclear significance. Correlation with tumor p53 mutation status is ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Garrick Wallstrom, Joshua LaBaer, Michael A. Hollingsworth, Daniel W. Cramer, Karen S. Anderson. Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 881. doi:10.1158/1538-7445.AM2014-881

Background: Chemoresistance is the major obstacle for the success of cancer treatment. The advanced breast cancer (BC) is commonly pan-resistant to different anticancer drugs. BC contains a small population of cancer stem cells (CSCs). CSCs express stem cell markers (CD24low/CD44high, ALDH+, CD133+) and are resistant to a wide range of anticancer drugs. Therefore, CSCs may be responsible for the pan-chemoresistance in advanced BC. We have recently developed several acquired chemoresistant BC cell lines. In this study, we investigated CSC traits in these cell lines and examined the cross-resistance of these cell lines to a panel of anticancer drugs. Disulfiram (DS), an antialcoholism drug, shows high cytotoxicity in several different types of cancer cell lines in vitro. It also targets CSCs and reverses pan-chemoresistance. However, the clinical application of DS in cancer treatment is limited by its very short half-life in the bloodstream. In order to increase the bio-stability of DS in the bloodstream, we recently developed a pluronic micelle-encapsulated DS (PM-DS). In this study, we examined the cytotoxicity of a newly developed (PM-DS) in the resistant cell lines. The effect of PM-DS on CSC traits in the resistant BC cell lines was also examined. Cell lines and Methods: Cell lines: MDA-MB-231, MCF7 and T47D and acquired resistant BC cell lines [gemcitabine (MDA-MB-231GEM100), 5-fluouracil (MCF7FU10) and doxorubicin (T47DDOX100nM)]; Methodologies: MTT assay, Western blot, CSC markers detection (ALDH, CD24low/CD44high, CD133), embryonic stem cell markers detection (Nanog, Sox2, Oct 4). Results: The resistant cell lines are highly cross-resistant to 6 conventional anticancer drugs e.g doxorubicin, gemcitabine, docetaxel, cisplatin, 5-fluouracil and vincristine. In comparison with the parental cells, the resistant cell lines have significantly longer doubling time. Significantly higher population of resistant cells expresses high levels of CSC markers e.g. ALDH+, CD24low/CD44high, and CD133+ and embryonic stem cell markers (OCT4, Sox2 and Nanog). DS was successfully encapsulated in pluronic micelles with a high entrapment efficiency of about 90% and our in vitro release studies showed that DS was slowly released from the micelles. Our formulation PM-DS has a relatively small size of about 200nm with high drug loading content (90μgDS/1mgPM). PM-DS was highly toxic to chemoresistant cell lines. It also targeted CSC population and enhanced cytotoxicity of anticancer drugs in the resistant cell lines. Conclusions: The acquired resistant BC cell lines are highly cross-resistant to 6 different kinds of anticancer drugs. CSCs may be responsible for the cross-chemoresistance. PM-DS targets CSC population and reverses the acquired pan-chemoresistance in acquired resistant BC cell lines. Key words: Disulfiram, micelles, breast cancer, stem-like cells. Citation Format: EREBI P. TAWARI, Peng Liu, Zhipeng Wang, Vinodh Kannappan, Christopher Mcconville, Angel Armesilla, John Darling, Juan Irache, Krassimira Yoncheva, Weiguang Wang, Petar Petrov. Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2015-4067

IGF-1R is frequently up-regulated in cancers including melanoma, and is implicated in mediating resistance to chemotherapy and targeted agents via regulation of apoptosis. Here, we investigated responses to temozolomide (TMZ), a methylating agent that generates toxic O6-methylguanine (O6meG) adducts, which are removed by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6meG adducts trigger futile cycles of mismatch repair, ultimately leading to formation of replication-associated DSBs. In a first approach to test links between IGF-1R and response to TMZ, we used a panel of 10 human melanoma cell lines in which we characterized MGMT expression, activation and expression of IGF axis components, and growth inhibitory effects of TMZ. We found that TMZ GI50 values correlated with MGMT levels (r=0.79, p=0.009), and in 7 MGMT-proficient cell lines, with phospho-IGF-1R (r=0.81, p=0.038) suggesting a link between TMZ resistance and IGF-1R activation. In a second approach we used IGF-1R tyrosine kinase inhibitors (TKIs) AZ12253801 and linsitinib (OSI-906), which is currently undergoing clinical evaluation, to assess effects of blocking IGF signaling. These IGF-1R TKIs were capable of sensitizing wild-type and mutant BRAF melanoma cells to TMZ; lack of correlation with apoptosis induction suggested that other factors contributed to this effect. IGF-1R inhibition did not influence MGMT expression or activity, was not epistatic with MGMT inhibition, and TMZ-sensitized MGMT-null cells, suggesting that chemo-sensitization was MGMT-independent. We found that IGF-1R TKI sensitized melanoma cells to inhibition of poly(ADP ribose) polymerase (PARP), and influenced processing of TMZ-induced DSBs, with increased RPA focus formation within 24hr of TMZ application, and persistence of RAD51 foci at 72hr. These data suggest functional impairment of homologous recombination (HR)-mediated DSB repair. Compared with simultaneous or prior IGF-1R TKI, TMZ sensitization was greater when IGF-1R TKI followed TMZ, consistent with a model in which IGF-1R inhibitor pre-treatment reduces DSB yield. In mice bearing melanoma xenografts with a clinically relevant (V600E BRAF, wild-type p53) genotype, this sequential (TMZ→linsitinib) combination treatment was tolerable, and linsitinib achieved peak plasma levels comparable to clinical Cmax values. TMZ and linsitinib caused minor inhibition of tumor growth (gradient reduction of 13% and 25% respectively), while the combination caused supra-additive growth delay (72%) that was significantly different from control (p<0.01), TMZ (p<0.01) and linsitinib (p<0.05) groups. In summary, IGF-1R inhibition influences HR repair of TMZ-induced DSBs and sensitizes melanomas to TMZ using treatment schedules relevant to clinical practice. Citation Format: Roger N. Ramcharan, Tamara Aleksic, Shan Gao, Jordan Tanner, Nicholas Darvill, Esther Bridges, Ruth Asher, Amanda J. Watson, Geoffrey P. Margison, Emmanouela Repapi, Ji-Liang Li, Mark R. Middleton, Valentine M. Macaulay. Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2014-1741

Goniothalamin (GTN) is a secondary metabolite styryl lactone isolated from several species of the tropical medicinal tree Goniothalamus. GTN has been shown to be cytotoxic and induce apoptosis in many cancer cell lines. This study sought to define the molecular basis underlying the antiproliferative actions of GTN. We synthesized the R and S enantiomers of GTN and found the R form to be more cytotoxic against a panel of breast cancer and lung cancer cell lines. The IC50 of GTN against breast cancer cell lines was in the range of 10-20 μM and interestingly, the SKBR3 cells, which harbor a R175H mutation in p53 were more sensitive to the drug. In contrast, the normal breast epithelial MCF10A cells were not killed by GTN, and N-acetylcysteine prevented cell-killing, indicating the ROS involvement. Indeed, the flow cytometry and cell staining with DCF-DA showed high levels of ROS generation, and this was accompanied by significant S-glutathionylation of bulk proteins. Other studies showed that GTN forms conjugate with glutathione with ease and deplete GSH levels in cells. Because p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by GTN may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. We also engineered the p53-null H1299 lung cancer cells to stably express the R175H mutant protein by lentiviral technology as an isogenic model. The conformation-specific antibodies for p53, namely Pab1620 that recognize the wt-p53 and pab420 that detects the mutant p53 were used for validating the p53 restoration. Immunoprecipitation and immunostaining using confocal microscopy showed that GTN treatment of R175H p53-containing cells results in a reciprocal loss of mutant protein and increase of wt-like protein. Further, the EMSA revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes. The changes were associated with a G2/M arrest and significant apoptosis. Increased levels of apoptotic markers suggested a priming action of GTN on cell death pathways. GTN also suppressed the SKBR3 cell migration and invasion at 5-10 μM. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay with either GTN alone or in combination with cisplatin. Our results shed light on the multiple mechanisms, including glutathione depletion, generation of redox imbalance, protein glutathionylation and p53 reactivation in the GTN cytotoxicity. We suggest that GTN-induced oxidative milieu facilitates a functional restoration of the mutant p53 through a thiolation of the redox-sensitive cysteines present in the DNA-binding domain. Our study will help to establish redox-perturbation as a paradigm for reactivation of the defective tumor suppressor (supported by a CPRIT grant [RP130266] to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Debasish Basak, Kalkunte S. Srivenugopal. Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3694.

Endocrine therapies using anti-estrogens are less toxic and very effective for breast cancers however tumor resistance to tamoxifen remains a stumbling block for successful therapy. Based on our recent study on the involvement of the DNA repair protein MGMT in pancreatic cancer (Clin Cancer Res. 15, 6087, 2009), we investigated whether MGMT overexpression mediates tamoxifen resistance. Specifically, we determined whether administration of MGMT inhibitor [O6-benzylguanine (BG)] at a non-toxic dose alone or in combination with the anti-estrogens (tamoxifen/fulvestrant) curtails human tamoxifen resistant breast cancer cell growth. Furthermore, we also determined whether BG sensitizes breast cancer cells to tamoxifen using tamoxifen resistant cells. MGMT expression was found to be increased in breast cancer cells relative to normal breast epithelial cells. Also, MGMT levels were significantly higher in tamoxifen resistant MCF-7 compared to the parent cells. Silencing of the ER-α expression using a specific siRNA resulted in augmentation of MGMT mRNA and protein levels by two fold. We also observed an inverse correlation between MGMT and p53 levels in breast cancer cell lines; moreover, p53 downregulation was accompanied by increased MGMT expression. Other experiments showed that BG alone or BG in combination with tamoxifen or fulvestrant decreased ER-α expression, whereas tamoxifen alone and fulvestrant alone increased and decreased the same respectively. All these treatments increased the p21cip1 mRNA and protein expression significantly. BG inhibited tamoxifen resistant breast cancer growth in a dose-dependent manner and it also resensitized resistant breast cancer cells to anti-estrogen therapy (TAM/ICI). These combinations also enhanced the cytochrome C release and PARP cleavage, indicative of apoptosis. In breast cancer xenografts, BG alone or a combination of BG with tamoxifen or fulvestrant caused a significant tumor growth delay and immunohistochemistry revealed that BG inhibited the expression of MGMT, ER- α, ki-67 and increased p21cip1 staining. These findings suggest that MGMT inhibition may provide a novel and effective approach for overcoming tamoxifen resistance (supported by a Florida Biomedical grant to SK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2011-619