Lachelle D. Weeks

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.

Uracil–DNA Glycosylase Expression Determines Human Lung Cancer Cell Sensitivity to Pemetrexed

Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthase. The base excision repair (BER) enzyme uracil–DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG−/− human colon cancer cells. Here, we examined the relationship between UNG expression and pemetrexed sensitivity in human lung cancer. We observed a spectrum of UNG expression in human lung cancer cells. Higher levels of UNG are associated with pemetrexed resistance and are present in cell lines derived from pemetrexed-resistant histologic subtypes (small cell and squamous cell carcinoma). Acute pemetrexed exposure induces UNG protein and mRNA, consistent with upregulation of uracil–DNA repair machinery. Chronic exposure of H1299 adenocarcinoma cells to increasing pemetrexed concentrations established drug-resistant sublines. Significant induction of UNG protein confirmed upregulation of BER as a feature of acquired pemetrexed resistance. Cotreatment with the BER inhibitor methoxyamine overrides pemetrexed resistance in chronically exposed cells, underscoring the use of BER-directed therapeutics to offset acquired drug resistance. Expression of UNG-directed siRNA and shRNA enhanced sensitivity in A549 and H1975 cells, and in drug-resistant sublines, confirming that UNG upregulation is protective. In human lung cancer, UNG deficiency is associated with pemetrexed-induced retention of uracil in DNA that destabilizes DNA replication forks resulting in DNA double-strand breaks and cell death. Thus, in experimental models, UNG is a critical mediator of pemetrexed sensitivity that warrants evaluation to determine clinical value. Mol Cancer Ther; 12(10); 2248–60. ©2013 AACR.

Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.

Radiosensitization of non-small-cell lung cancer cells and xenografts by the interactive effects of pemetrexed and methoxyamine

BACKGROUND AND PURPOSE The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We tested whether methoxyamine enhances pemetrexed-mediated radiosensitization of lung adenocarcinoma cells and xenografts. MATERIALS AND METHODS A549 and H1299 cells were evaluated for cell cycle distribution by flow cytometry, radiosensitization by clonogenic assay, and DNA repair by neutral comet assay and repair protein activation. H460 cells were included in some studies. Xenografts in nude mice received drug(s) and/or radiation, and tumor growth was monitored by caliper and in vivo toxicity by animal weight. RESULTS Exposure to pemetrexed/methoxyamine for 24 (H1299, H460) or 48 (A549)hours before irradiation resulted in accumulation of cells near the radiosensitive G1/S border; dose-enhancement factors of 1.62±0.19, 1.97±0.25, and 1.67±0.30, respectively; reduction of mean inactivation dose by 32%, 30%, and 46%, respectively; and significant reductions of SF2 and SF4 (p<0.05). Radiosensitization was associated with rapid DNA double-strand-break rejoining and increased levels of DNA-PKcs. Both tumor-growth rate and tumor-growth delay were significantly improved by adding methoxyamine to pemetrexed pre-irradiation (p<0.0001); no mice lost weight during treatment. CONCLUSIONS Addition of methoxyamine to pemetrexed and fractionated radiotherapy may improve outcome for patients with locally advanced non-squamous non-small-cell lung cancer.

Abstract 4696: Expression of the DNA repair protein UNG predicts lung cancer sensitivity to pemetrexed

Uracil misincorporation is a direct consequence of TS inhibition by the multi-target antifolate, pemetrexed. The DNA base excision repair (BER) enzyme Uracil DNA glycosylase (UNG) is the major glycosylase responsible for the removal of misincorporated genomic uracil. We have previously illustrated that human colon cancer cells (DLD1) with engineered stable somatic knockout of UNG expression are hypersensitive to pemetrexed owing to the cytotoxicity of genomic uracil accumulation. Here, we examined the relationship between the expression of UNG and the pemetrexed sensitivity in a small panel of lung cancer cell lines (n∼10). The levels of UNG were measured by western blot (for protein) and RT-PCR (for mRNA). Results revealed that UNG mRNA expression is well correlated with protein expression in all cell lines. Moreover, there was a wide range of distribution of UNG levels in the lung cancer cell lines evaluated. UNG cutting assay and AP site measurements confirm that these differences in UNG expression result in varied potential for BER activation and subsequent AP site formation. To determine the role of this variation in UNG expression in pemetrexed cytotoxicity, colony survival studies were performed. In general, cell lines with higher UNG expression were more resistant to pemetrexed. H460 cells having 7-fold lower expression of UNG mRNA than H1975 cells are >5 fold more sensitive to pemetrexed than H1975 cells (H460 IC50 = 200nM; H1975 IC50 = >1000nM). Analysis of tissue microarray data in Oncomine confirms variations in UNG expression in primary human lung cancer samples. Moreover, small cell lung cancer and squamous cell lung cancer, both notoriously resistant to pemetrexed, have significantly increased median UNG expression compared to all other lung cancer subtypes (1.5-fold greater UNG expression p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4696. doi:1538-7445.AM2012-4696

Abstract 4484: Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase (TS). The DNA base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG-/- human colon cancer cells (DLD1) and proposed that cytotoxic uracil accumulation leads to collapsed replication forks, double strand breaks and cell death. Here, we examined the relationship between cellular capability to remove uracil-DNA and pemetrexed sensitivity in human lung cancer cells. Viral siRNA and shRNA knockdown of UNG enhanced pemetrexed sensitivity in A549 and H1975 cells, respectively. We further evaluated induction of UNG as a mechanism of pemetrexed resistance. Exposure of H1299 cells to step-wise increasing concentrations of pemetrexed established drug resistant sublines: H1299PR-1 and H1299PR-2. Significant induction of UNG protein was observed in both sublines. Importantly, co-treatment with the BER inhibitor, methoxyamine (MX), overcame pemetrexed resistance in H1299PR-1/2 underscoring the utility of BER directed therapeutics to offset acquired pemetrexed resistance. Lastly, differential expression of UNG in cell lines and primary lung cancer tissues was associated with pemetrexed sensitivity. Cell lines derived from squamous and small cell carcinoma, known to be clinically resistant to pemetrexed, had the highest levels of UNG expression in a panel of eight lung cancer cell lines. Analysis of cDNA microarrays derived from primary human lung cancer tissues also indicated high UNG expression in pemetrexed-resistant histological subtypes. Thus, we propose that UNG is a major determinant of pemetrexed sensitivity in human lung cancer. Citation Format: Lachelle D. Weeks, Pingfu Fu, Lili Liu, Stanton L. Gerson. Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed. [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 4484. doi:10.1158/1538-7445.AM2013-4484

Abstract 4482: Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Misincorporation of genomic uracil and formation of DNA double strand breaks are known consequences of exposure to TS inhibitors such as 5-fluorouracil, and pemetrexed. Uracil DNA glycosylase catalyzes the excision of genomic uracil and initiates DNA base excision repair (BER). Thus, a relationship between antifolate cytotoxicity and UNG expression and activity has been hypothesized. However, a precise mechanism linking antifolate-induced formation of DSBs to genomic uracil accumulation and UNG-initiated BER has not been described. Herein, we report that despite equivalent proliferation indices, DLD1 UNG-/- cells are more sensitive to pemetrexed mediated intra S-phase arrest, DNA double strand break formation and apoptosis compared to UNG+/+ cells. Using data from western blots in chromatin extracts, PCNA staining of cells in S-phase, and pulse-chase labeling of replicating cells with CldU and IdU, we surmise that the accumulation of uracil in pemetrexed-treated UNG-/- cells is associated with significant replication fork instability. In addition, UNG-/- cells have reduced capacity to recover from pemetrexed-mediated DNA damage, as indicated by the persistence of S-phase arrest and gamma-H2AX foci. This defect in recovery was not explained by double strand break repair capacity, which was equivalent in UNG+/+ and UNG-/- cells. Using γ-H2AX ChIP sequencing, we observed a 5-fold increase in the number of γ-H2AX binding sites in UNG-/- cells compared to UNG+/+ cells treated at IC50 levels of pemetrexed. This analysis evinced distinct patterns of γ-H2AX binding in UNG+/+ and UNG-/- cells. Double strand breaks (γ-H2AX) were more significantly associated with transcription start sites and putative origins of replication in UNG-/- cells compared to UNG+/+ cells. Based on these data we conclude that uracil accumulation, and thus UNG activity, during pemetrexed exposure directs both the quantity and the location of double strand breaks. These findings support uracil mediated S-phase arrest and DNA replication fork collapse as the mechanism of double strand break formation and cell death in pemetrexed treated UNG-/- cells. Citation Format: Lachelle D. Weeks, Gabriel Zentner, Peter Scacheri, Stanton L. Gerson. Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer 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 4482. doi:10.1158/1538-7445.AM2013-4482

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 A100: Pemetrexed-induced uracil accumulation is an importatn source of DNA damage in UNG deficient cells, contributing to hematologic toxicity.

Pemetrexed is a multi-target antifolate that has proven activity against several solid tumors. Dose limiting myelosuppression and acquired resistance are major challenges to pemetrexed and antifolate chemotherapy regimens. We have previously shown that resistance to pemetrexed in human cancer cells can be overcome via inhibition of DNA base excision repair (BER), the major pathway responsible for the resolution of uracil in DNA. To understand the implications of UNG deficiency in vivo, we have investigated the impact of UNG deficiency on whole animal response to pemetrexed. Here, we illustrate that pemetrexed treatment induces more significant reduction in bone marrow cell numbers and relatively low peripheral lymphocyte counts in UNG−/− mice compared to UNG+/+ littermates. Compared to wild-type cells, UNG−/− MEFs also show reduced viability and increased cell death in response to pemetrexed. Hematopoietic cell sensitivity was mirrored by increased pemetrexed cell killing in UNG mutant human lymphoblastoid cells, derived from patients with biallelic inactivating mutations in the UNG gene. Cellular sensitivity is characterized by reduced nucleotide incorporation, arrest in S-phase of the cell cycle, and induction of apoptosis. Pemetrexed treatment of UNG−/− cells also causes robust induction of γ-H2AX, a surrogate marker of DNA double strand breaks. Through γ-H2AX ChIP-sequencing we provide the first direct evidence that DNA double strand breaks are located specifically at AT(U) rich sequences, directly implicating uracil accumulation in the formation of double strand breaks following pemetrexed treatment. UNG deficient cells showed no differential sensitivity to cisplatin or temozolomide, two drugs unrelated to uracil accumulation. Moreover, through thymidine supplementation, we were able to partially reverse the pemetrexed-mediated induction of DNA damage markers, including γ-H2AX and protect UNG−/− cells from cell death, further supporting a uracil-mediated mechanism of cytotoxicity in UNG−/− cells. In sum, these data suggest that the accumulation of uracil in the DNA of hematopoietic cells drives myelosuppression and lymphocyte toxicity. Mechanistically, our data support a previously underappreciated role for uracil accumulation, UNG activity and the BER pathway in the determination of cellular sensitivity to pemetrexed and suggest UNG expression may be an important predictor of adverse events, such as myelosuppression in individuals receiving pemetrexed. 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 A100.

Abstract 5490: Mice deficient in uracil DNA glycosylase (UDG) display increased sensitivity to the antifolate pemetrexed

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pemetrexed, a multi-target antifolate, causes genomic uracil incorporation through inhibition of the enzymes thymidylate synthetase (TS) and dihydrofolate reductase (DHFR). Base excision repair (BER), initiated by uracil DNA glycosylase (UDG), actively recognizes and removes misincorporated uracil from the genome. Mice deficient in UDG have been described and display increased genomic uracil accumulation, elevated spontaneous mutation frequency, and higher incidence of B-cell lymphoma. We have previously reported the in vitro induction of UDG in H460 human lung cancer cells after pemetrexed exposure and have illustrated hypersensitivity to pemetrexed in human colon cancer cell lines lacking UDG expression (DLD1 UDG-/-). Here, we have investigated the role of UDG expression on pemetrexed sensitivity in vivo using gene-targeted UDG-/- mice. Mice received either a single 150mg/kg dose of pemetrexed on day 1 or received 3 consecutive 150 mg/kg doses of pemetrexed on days 1-3 and were sacrificed on day 4 or 5, respectively. Compared to UDG+/+ and UDG+/- littermates, UDG-/- mice were markedly myelosuppressed following pemetrexed treatment, as evidenced by a ∼50% reduction in bone marrow cell numbers and a ∼70% reduction in total colony forming units (CFU). Moreover, marrow from pemetrexed-treated UDG-/- mice expressed higher levels of DNA damage markers γ-H2AX and cleaved-PARP than control samples suggesting the formation of DNA double strand breaks and activation of apoptosis in UDG-/- marrow following pemetrexed exposure. Western blot analysis further evinced Chk-1 phosphorylation (p-Chk1, Ser345) in marrow from UDG-/- mice but not UDG+/+ or UDG+/- samples, suggesting inadequate removal of pemetrexed-induced genomic uracil may stall DNA replication and contribute to the cytotoxicity observed. These data in addition with our novel observations of enhanced pemetrexed sensitivity in UDG-/- mouse embryonic fibroblast (MEFs), UDG mutant human lymphoblastoid cell lines (LCLs), and DLD1 UDG-/- cells, strongly suggest that UDG deficiency potentiates pemetrexed cytotoxicity in in vitro as well as in in vivo models. Additionally, our findings indicate a role for genomic uracil accumulation in the mechanism of cytotoxicity for pemetrexed. Furthermore, our data illustrate a synthetic lethal interaction between TS/DHFR inhibition and UDG deficiency and promote UDG as a target for improving pemetrexed efficacy. 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 5490. doi:10.1158/1538-7445.AM2011-5490