Alina Bulgar

Combined Treatment with Temozolomide and Methoxyamine: Blocking Apurininc/Pyrimidinic Site Repair Coupled with Targeting Topoisomerase IIα

Purpose: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models. We postulate that the enhanced cytotoxicity is mediated by methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIα (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. Experimental Design: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease– and topo II–mediated DNA cleavage in vitro. Results: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II–mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of γH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by γH2AX foci are associated with topo II in cells. Conclusions: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.

Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair.

Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due to the rapid removal of uracil from DNA by UDG and subsequent repair of the resultant AP sites (abasic sites) via the base excision repair (BER). The resistance to pemetrexed in UDG+/+ cells could be reversed by the addition of methoxyamine (MX), which binds to AP sites and interrupts BER pathway. Furthermore, MX-bound AP sites induced cell death was related to their cytotoxic effect of dual inactivation of UDG and topoisomerase IIα, two genes that are highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more selective cytotoxicity on cancer cells through a synthetic lethal mechanism.

Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair

Use of chemotherapeutic agents to induce cytotoxic DNA damage and programmed cell death is a key strategy in cancer treatments. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway resulted in formation of abasic or apurinic/apyrimidinic (AP) sites, and blockage of AP sites led to a significant enhancement of drug sensitivity due to reduction of DNA base excision repair. Since a number of chemotherapeutic agents also induce formation of AP sites, monitoring of these sites as a clinical correlate of drug effect will provide a useful tool in the development of DNA-targeted chemotherapies aimed at blocking abasic sites from repair. Here we report an imaging technique based on positron emission tomography (PET) that allows for direct quantification of AP sites in vivo. For this purpose, positron-emitting carbon-11 has been incorporated into methoxyamine ([(11)C]MX) that binds covalently to AP sites with high specificity. The binding specificity of [(11)C]MX for AP sites was demonstrated by in vivo blocking experiments. Using [(11)C]MX as a radiotracer, animal PET studies have been conducted in melanoma and glioma xenografts for quantification of AP sites. Following induction of AP sites by temozolomide, both tumor models showed significant increase of [(11)C]MX uptake in tumor regions in terms of radioactivity concentration as a function of time, which correlates well with conventional aldehyde reactive probe (ARP)-based bioassays for AP sites.

Abstract 682: Dual inhibition of BER by TRC102 and PARP inhibitor (ABT 888) synergistically enhances cytotoxicity of TMZ in human melanoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Melanoma, the most fatal skin cancer, has increased in incidence by 15-fold in the past 40 years, the fastest rate increase of any human malignancy. This disease metastasizes rapidly and is highly resistant to chemotherapy. Currently, temozolomide (TMZ) is an important part of treatment regimens for advanced metastatic melanoma. However, drug resistance often results in treatment failure. A major resistance factor is the presence of elaborate mechanisms of DNA repair. We have previously studied the role of base excision repair (BER) in conferring TMZ resistance and explored a combined therapy by targeting BER with methoxyamine (TRC102), an inhibitor of BER, which binds to abasic sites and disrupts the BER pathway. The potentiation of TMZ by TRC102 has been validated in different tumor types in vitro and in xenograft models. Results showed TRC102 potentiation of TMZ was accompanied by a remarkable induction of DNA strand breaks. In this study, we examined the therapeutic efficacy of the combination of TMZ with TRC102 and a PARP inhibitor (ABT888) in three melanoma cell lines (A375, WM9, and WM164) and in melanoma xenografts. PARP, an enzyme critical in BER pathway, is actively involved in the repair of DNA strand breaks. We hypothesized the inhibition of PARP would inhibit BER with the accumulation of large numbers of unrepaired DNA strand breaks and the combination of TRC102 and ABT888 would synergistically enhance the anti-tumor effect of TMZ through dual inhibition of BER. Results showed that compared to TMZ alone, the combination with either ABT888 or TRC102 more effectively inhibited cell viability and induced apoptosis in A375 and WM9, but not in WM164 melanoma cell lines. The resistance in WM164 cells is probably related to a deficiency in methylpurine-DNA glycosylase, which is responsible for removing TMZ-induced methylated DNA adducts (N7mG and N3mA) and producing toxic AP sites. When combined with TMZ and TRC102, ABT888 at a concentration of 5 µM efficiently potentiated cytotoxicity by 8-10 fold in A375 and WM9 melanoma cell lines, respectively, and 4 fold in WM164 cells. Cytotoxicity was correlated with the induction of DNA single and double stranded breaks as assayed by comet assay and induction of γ-H2AX. For in vivo studies, the therapeutic regimen was initiated when tumor xenografts (WM9) in nude mice reached ∼100 mm3 in volume and treatment was continued for 5 days. Tumor volume was measured for assessment of therapeutic effect. At termination, we found 30-40% reduction in tumor volume following treatment with the combination of TMZ with ABT888 or with TRC2, and 80% reduction in tumor volume by combining the three drugs relative to the TMZ alone group, which had no significant differences in tumor growth in comparison to untreated group. Data indicate that combining a PARP inhibitor with TMZ and TRC102 results in greater inhibition of BER and induces a synergistic cytotoxic effect. 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 682.

Abstract A104: Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: Overriding the limit with methoxyamine (TRC102) to inhibit base excision repair.

Uracil DNA glycosylase (UDG) is a conserved DNA repair protein existing in all types of human cells. Since UDG specifically removes uracil bases from DNA, its repair activity determines cell sensitivity to anticancer agents that are capable of introducing uracil into DNA. We studied the participation of UDG in response to pemetrexed-induced incorporation of uracil into DNA using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). Results revealed that UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. In contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due to a rapid removal of uracil from DNA, generating abasic sites (AP sites). Resultant AP sites were efficiently repaired by base excision repair (BER) so that they contributed little to cell death. However, addition of the BER inhibitor methoxyamine (TRC102) synergistically enhanced the cytotoxicity of pemetrexed in UDG proficient cells. MX bound covalently to AP sites to form unrepairable MX-bound AP sites, thereby inhibiting the BER pathway. Importantly, we identified that MX-bound AP sites induced cell death by inactivating UDG and poisoning topoisomerase II. These two genes are highly expressed in lung cancer cells in comparison with normal bone marrow cells. Thus, targeting UDG/BER to enhance pemetrexed cytotoxic effects is a target-based strategy that selectively mediates cytotoxicity on cancer cells through a synthetic lethal mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A104.

Abstract LB-138: In vivo imaging of DNA damage and repair

Objectives: To directly quantify abasic sites induced by DNA-targeted chemotherapeutic agents for in vivo studies of base excision repair pathway. Background: Current cancer treatments rely heavily on chemotherapeutic agents to induce cytotoxic DNA damages and programmed cell death in ancer cells. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway results in formation of abasic or apurinic/apyrimidinic (AP) sites and inhibition of AP sites leads to significant reduction of drug resistance and enhancement of drug sensitivity. Thus, AP-site formation has been identified as an important biomarker in DNA-targeted chemotherapies. Methods: Design, synthesis, and evaluation of PET imaging agents that bind to AP sites with high affinity and specificity. We date, we have developed positron-emitting [ 11 C]methoxyamine for positron emission tomography (PET) that allows for quantification of AP sites in vivo. Results: [ 11 C]methoxyamine has been synthesized with high radiochemical yield and purity. Following radiolabelling, microPET studies have been conducted to evaluate their pharmacokinetic profiles in melanoma and glioma xenograft tumor mouse models that are pre-treated with temozolomide to induce AP-site formation. Subsequent quantitative analysis showed that the radioactivity concentration were elevated in proportion to the AP sites induced in tumor regions pre-treated with temozolomide relative to tumor regions without any treatment. In vivo blocking studies based on microPET also showed that the agents bound to AP sites with high specificity. Conclusion : our studies demonstrated that PET imaging can be used to monitor BER pathway and evaluate efficacy of DNA-targeted therapeutic treatments in cancer. 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 LB-138.