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Prevention of Base Excision Repair by TRC102 (Methoxyamine) Potentiates the Anti-Tumor Activity of Pemetrexed in Vitro and in Vivo L

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Prevention of Base Excision Repair by TRC102 (Methoxyamine) Potentiates the Anti-Tumor Activity of Pemetrexed in Vitro and in Vivo L Prevention of Base Excision Repair by TRC102 (Methoxyamine) Potentiates the Anti-Tumor Activity of Pemetrexed in vitro and in vivo L. Liu, A. Bulgar, J. Donze, B. Adams, C.P. Theuer, S.L. Gerson Case Comprehensive Cancer Center, Cleveland, Ohio and TRACON Pharmaceuticals ABSTRACT RESULTS PURPOSE: TRC102 (methoxyamine) reverses resistance to alkylating agents by AP Sites Detected in H460 Cells inhibiting base excision repair (BER; a mechanism of DNA repair), thereby increasing TRC102 Bound AP Sites are Refractory to the Repair The Combination of Pemetrexed and TRC102 Enhances After Treatment with Pemetrexed and TRC102 DNA strand breaks and potentiating the anti-tumor activity of alkylating agents without by AP Endonuclease DNA Double Strand Breaks and Apoptosis additional toxicity. Based on these data, TRC102 is currently being studied in that is Independent of the Bcl2 Pathway Pemetrexed A. B. combination with temozolomide in a Phase 1 trial. We hypothesized that nucleotide 35 14 Control APE Pemetrexed + TRC102 (6 mM) Pemetrexed (200 µM) imbalances caused by the anti-folate pemetrexed would also produce AP sites 30 12 Pemetrexed+TRC102 + - + - recognized and repaired by BER, and that inhibition of BER by TRC102 would TRC102 (6 mM) C P P+T P P+T P P+T T 25 10 U therefore improve the anti-tumor activity of pemetrexed. METHODS: Pemetrexed- UDG 20 8 40 mer Cleaved PARP induced apurinic/apyrimidinic (AP) sites and BER inhibition were quantified using an AP Site 15 6 AP site assay in vitro. Single and double DNA strand breaks were quantified by the TRC102 20 mer H2AX Comet assay in vitro, apoptosis was quantified by Annexin V staining, and anti-tumor 10 4 AP sites (arbitrary unit) sites (arbitrary AP AP sites (arbitrary unit) sites (arbitrary AP Bax TRC102-AP Site activity was assessed in an in vivo xenograft study of subcutaneously implanted 5 2 AP Site TRC102-AP Site human lung, colorectal and breast cancer cells. RESULTS: AP sites increased Bcl-2 0 0 proportionally with dose in pemetrexed–treated H460 lung cancer cells in vitro. 0 100 200 400 24 48 72 Tubulin TRC102 reduced the number of available AP sites in pemetrexed-treated cells (by 60- Pemetrexed (µM) Time (hrs) A. Schematic diagram indicate the preparation of a position specific oligonucleotide 80%), indicating successful inhibition of BER. TRC102 treatment increased DNA substrates containing an AP site or an TRC102 bound AP site. BER Protein Levels in H460 Cells B. APE has the ability to cleave the AP site but not the TRC102 bound AP site, P 100 µM P 200 µM P 400 µM strand breaks (2 fold increase versus treatment with pemetrexed alone) and apoptosis. Before and After Treatment with Pemetrexed or the Combination indicating that TRC102 bound AP site is refractory to the repair by APE. TRC102 increased the activity of pemetrexed in vivo (tumor growth delay of 2 days in Cells were treated with TRC102 at 6 mM and collected at 24 hr mice bearing H460 or A549 lung cancer xenografts treated with 150mg/kg C P P+T P P+T P P+T T pemetrexed alone versus 9 days in mice treated with 150mg/kg pemetrexed + 4mg/kg UDG TRC102 Enhances Antitumor Effect of Pemetrexed in Nude Mice Carrying Human Tumors TRC102; p<0.05); in vivo systemic toxicity was not increased and TRC102 alone had C = Control no effect in vitro or in vivo. TRC102 also increased pemetrexed activity on HCT116 FEN1 T = TRC102 H460 A549 colorectal and MDA-MB-468 breast cancer xenografts. Moreover, the combination P = Pemetrexed β-pol 2500 2500 selectively up-regulated the BER proteins, uracil DNA glycosylase and polymerase β, APE Control Control ) ) 3 which provides strong evidence that DNA damage induced by the drug combination 3 2000 2000 Pemetrexed Pemetrexed induces BER. CONCLUSION: TRC102 effectively inhibits BER in cancer cells treated Tubulin Pemetrexed +TRC102 Pemetrexed+TRC102 TRC102 with pemetrexed. Inhibition of BER by TRC102 results in an increase in DNA strand 1500 1500 TRC102 breaks and apoptosis, and improved anti-tumor activity versus treatment with P 100 µM P 200 µM P 400 µM 1000 1000 pemetrexed alone. Given its preclinical safety and efficacy profile, clinical study of Cells were treated with TRC102 at 6 mM and collected at 24 hr TRC102 combined with pemetrexed is warranted. Tumor volume(mm Tumor volume(mm * 500 * 500 Base Excision Repair Pathway 0 0 Neutral Comet 0246810121416 0 5 10 15 20 25 30 Days Days Base Excision Glycosylase * P < 0.05 Repair pathway HCT116 MDA-MB-468 AP site 1800 800 PARP Alkali Comet 1600 Control 700 Control pol β Pemetrexed Pemetrexed ) 1400 APE 3 600 Pemetrexed + TRC102 ) Pemetrexed+ TRC102 3 1200 TRC102 Pemetrexed Pemetrexed+TRC102 TRC102 500 TRC102 1000 PNCA 400 pol β Polδ/ε 800 * DNA Strand Breaks Measured by Comet Assay 300 600 in H460 Cells 4 hours after Treatment 200 Tumor volume(mm 400 Tumor volume(mm * 100 pol β XRCC1 200 FEN1 Alkaline comet assay Neutral comet assay 0.8 1.2 0 0 0 10 20 30 40 50 60 70 80 90 100 0.7 0 5 10 15 20 25 LigIII 1 Days Days * P < 0.05 Lig I Lig I 0.6 0.5 0.8 0.4 0.6 SUMMARY 0.3 0.4 Hypotheses: (arbitrary units) (arbitrary units) 1) Pemetrexed induces the incorporation of abnormal bases that are removed by DNA Glycosylases as part of base excision repair (BER), 0.2 thereby producing AP sites. 1) Pemetrexed inhibits several key enzymes in the de novo pathways of pyrimidine 0.2 DNA single strand breaks and purine biosynthesis, leading to nucleotide pool imbalances, which favor the 0.1 breaks DNA strand double Incorporation of mismatched bases to initiate base excision repair (BER). 0 0 2) TRC102 binds to AP sites to efficiently block BER and increase DNA strand breaks. Control Pemetrexed P+T TRC102 Control Pemetrexed P+T TRC102 3) Inhibition of BER by TRC102 enhances pemetrexed antitumor activity against several human solid tumors, including lung, breast and 2) TRC102 blocks BER and enhances cytotoxicity of pemetrexed. colorectal cancers..
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