oxaliplatin-DNA Adducts Research Articles

Oxaliplatin-DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy.

FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 μmol/L) or diagnostic microdose (1 μmol/L) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [14C]oxaliplatin prior to treatment, as well as a second [14C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

Abstract CT055: Microdose induced oxaliplatin-DNA adducts as a predictive biomarker of response in colorectal cancer

Abstract Background Combination 5-FU and oxaliplatin (FOLFOX) is standard therapy for colorectal cancer (CRC) in neoadjuvant, adjuvant, and metastatic settings. However, oxaliplatin neuropathy is the main dose-limiting toxicity that often necessitates dose reduction or discontinuation before progression. Prediction of oxaliplatin sensitivity in tumor cells would be valuable for dose optimization to maximize anti-tumorigenic effect and limit sensory neuropathy. Oxaliplatin functions via the formation of covalent drug-DNA adducts which ultimately inhibit cell replication and promote apoptosis. Detection of these drug-DNA adducts could serve as a biomarker of oxaliplatin efficacy. We therefore hypothesized that drug-DNA adduct levels induced by sub-therapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are predictive of tumor responses. Methods The feasibility of this predictive diagnostic microdosing approach was assessed in CRC cell culture and a pilot clinical trial of six CRC patients. Four CRC cell lines were cultured with therapeutically relevant (100 µM) or diagnostic microdose (1 µM) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct levels with accelerator mass spectrometry, an ultrasensitive isotope analysis technique. Adduct formation was correlated with oxaliplatin cytotoxicity as measured by the MTT viability assay. In a pilot clinical trial (NCT02569723), six CRC patients received an intravenous diagnostic microdose containing [14C]oxaliplatin prior to treatment and a second [14C]oxaliplatin dose during FOLFOX chemotherapy. Adduct levels from peripheral blood mononuclear cells (PBMC), used as surrogates for tumor cells, were correlated to mean tumor volume change of evaluable target lesions. Results Drug-DNA adduct levels were linearly proportional between microdoses and therapeutically relevant concentrations in cell culture (R2 = 0.91, p < 0.0001) and patient samples (R2 = 0.63, p < 0.0001). Plasma pharmacokinetics of microdoses were consistent with those of therapeutic dosing. No microdose-associated toxicity was observed in this patient population. PBMC adduct levels significantly correlated with tumor shrinkage (microdose: R2 = 0.82, p = 0.033; therapeutic dose: R2=0.65, p = 0.099). Conclusion Diagnostic microdosing with oxaliplatin is feasible and is predictive of oxaliplatin sensitivity in CRC patients. Citation Format: Justin Chen, May Cho, Maike Zimmermann, Thomas Semrad, Chun-Yi Wu, Aiming Yu, Chong-Xian Pan, Paul T. Henderson, Edward J. Kim. Microdose induced oxaliplatin-DNA adducts as a predictive biomarker of response in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT055.

Structural basis for the bypass of the major oxaliplatin-DNA adducts by human DNA polymerase η.

Oxaliplatin, together with cisplatin, is among the most important drugs used in cancer chemotherapy. Oxaliplatin, which contains a bulky diaminocyclohexane (DACH) moiety, kills cancer cells mainly by producing (DACH)Pt-GpG intrastrand cross-links that impede transcription. The Pt-GpG tolerance by translesion DNA synthesis (TLS) polymerases contributes to the resistance of tumors to platinum-based chemotherapy. In particular, human DNA polymerase η (Polη) readily bypasses Pt-GpG adducts. While many structural studies have addressed how TLS polymerases interact with cisplatin-DNA adducts, a structure of DNA polymerase in complex with oxaliplatin-DNA adducts has not been reported, limiting our understanding of bypass of the bulky (DACH)Pt-GpG lesion by TLS polymerases. Herein, we report the first structure of DNA polymerase bound to oxaliplatinated DNA. We determined a crystal structure of Polη incorporating dCTP opposite the 3'G of the (DACH)Pt-GpG, which provides insights into accurate, efficient bypass of the oxaliplatin-GpG adducts by TLS polymerases. In the catalytic site of Polη, the 3'G of the (DACH)Pt-GpG formed three Watson-Crick hydrogen bonds with incoming dCTP and the primer terminus 3'-OH was optimally positioned for nucleotidyl transfer. To accommodate the bulky (DACH)Pt-GpG lesion, the Val59-Trp64 loop in the finger domain of Polη shifted from the positions observed in the corresponding Polη-cisplatin-GpG and undamaged structures, suggesting that the flexibility of the Val59-Trp64 loop allows the enzyme's bypass of the (DACH)Pt-GpG adducts. Overall, the Polη-oxaliplatin-GpG structure provides a structural basis for TLS-mediated bypass of the major oxaliplatin-DNA adducts and insights into resistance to platinum-based chemotherapy in humans.

Correlation of Platinum Cytotoxicity to Drug-DNA Adduct Levels in a Breast Cancer Cell Line Panel.

Platinum drugs, including carboplatin and oxaliplatin, are commonly used chemotherapy drugs that kill cancer cells by forming toxic drug-DNA adducts. These drugs have a proven, but modest, efficacy against several aggressive subtypes of breast cancer but also cause several side effects that can lead to the cessation of treatment. There is a clinical need to identify patients who will respond to platinum drugs in order to better inform clinical decision making. Diagnostic microdosing involves dosing patients or patient samples with subtherapeutic doses of radiolabeled platinum followed by measurement of platinum-DNA adducts in blood or tumor tissue and may be used to predict patient response. We exposed a panel of six breast cancer cell lines to 14C-labeled carboplatin or oxaliplatin at therapeutic and microdose (1% therapeutic dose) concentrations for a range of exposure lengths and isolated DNA from the cells. The DNA was converted to graphite, and measurement of radiocarbon due to platinum-DNA adduction was quantified via accelerator mass spectrometry (AMS). We observed a linear correlation in adduct levels between the microdose and therapeutic dose, and the level of platinum-DNA adducts corresponded to cell line drug sensitivity for both carboplatin and oxaliplatin. These results showed a clear separation in adduct levels between the sensitive and resistant groups of cell lines that could not be fully explained or predicted by changes in DNA repair rates or mutations in DNA repair genes. Further, we were able to quantitate oxaliplatin-DNA adducts in the blood and tumor tissue of a metastatic breast cancer patient. Together, these data support the use of diagnostic microdosing for predicting patient sensitivity to platinum. Future studies will be aimed at replicating this data in a clinical feasibility trial.

Abstract 905: Molecular dissection of platinum resistance through functional analysis

Abstract BACKGROUND: Platinum (Pt) agents (cisplatin, carboplatin and oxaliplatin) are active in many cancers including bladder cancer. Chemoresistance is the most common cause of treatment failure. This study is to determine the feasibility of using ultrasensitive accelerator mass spectrometry (AMS) to identify chemoresistance after cancer cells or patients are treated with one non-toxic microdose (1/100th of therapeutic dose) of Pt agents. The long-term goal is to identify chemoresistance before cancer patients receive toxic chemotherapy, and to determine the underlying resistance mechanisms to design personalized chemotherapy. METHODS: Cellular sensitivity to chemotherapeutic agents was determined by the MTT assay. Platinum-induced DNA adduct formation and repair of adducts was measured with AMS after cells were exposed 14C-labeled carboplatin and oxaliplatin. AMS quantifies the 14C label that is attached to genomic DNA when the 14C-labled drug forms adducts with DNA. Cell uptake and efflux was measured by liquid scintillation counting. Intracellular glutathione levels were measured by colorimetric analysis. RESULTS: Compared to the parental bladder cancer 5637 cells, chemoresistant 5637R cells are resistant to oxaliplatin (IC50: 2.45 µM versus 27.27 µM, p<0.0001), and cisplatin (0.59 µM versus 2.99 µM, p=0.049), carboplatin (24.34 µM versus 72.18 µM, p<0.0001), and gemcitabine (0.12 µM versus 1.44 µM, p=0.0015). Both 5637 and 5637R cells are still sensitive to other chemotherapeutic agents commonly used in treating bladder cancer, such as doxorubicin, methotrexate and vinblastine. Consistent with our hypothesis, chemoresistant 5637R cells have low oxaliplatin-induced DNA adduct levels than the parental 5637 cells (AUC of 943 versus 2,772 adducts per 109 nucleotide-hour for 5637, p=0.001). This low level of oxaliplatin-DNA adduct formation might be secondary to the pre-DNA damage mechanisms, such as decreased uptake (AUC of 4.42 versus 5.12 X 109 oxaliplatin molecules per cell for 5637, p=0.037) and increased intracellular inactivation of oxaliplatin by glutathione (53.91 versus 46.93 nmol/mg protein for 5637, p=0.003), plus increased repair of oxaliplatin-DNA adducts (3.48 versus 1.34 adducts per 108 nucleotides per hour for 5637, p=0.0004). We found the same correlation of low Pt-DNA adduct levels and chemoresistance in non-small cell lung (NSCLC) and breast cancer cell lines, and determined the same resistant mechanisms, such as cell uptake/efflux, intracellular inactivation and DNA repair. Carboplatin had partially different resistant mechanisms. CONCLUSION: Functional analysis of major resistant steps can identify some chemoresistance mechanisms that can potentially help design personalized chemotherapy to overcome resistance. This approach can be applied to several different cancer types. A Phase 0 microdosing clinical trial is currently going on in patients with NSCLC and bladder cancer. Citation Format: Amy W. Pan, Sisi Wang, Hongyong Zhang, Ruth Vinall, Tzu-yin Lin, Michael Malfatti, Maike Zimmermann, Tiffany Scharadin, Kenneth Turteltaub, Ralph de Vere White, Chong-xian Pan, Paul Henderson. Molecular dissection of platinum resistance through functional analysis. [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 905. doi:10.1158/1538-7445.AM2014-905

Glutathione modified CdTe quantum dots as a label for studying DNA interactions with platinum based cytostatics

Cisplatin, carboplatin, and oxaliplatin represent three generations of platinum based drugs applied successfully for cancer treatment. As a consequence of the employment of platinum based cytostatics in the cancer treatment, it became necessary to study the mechanism of their action. Current accepted opinion is the formation of Pt-DNA adducts, but the mechanism of their formation is still unclear. Nanomaterials, as a progressively developing branch, can offer a tool for studying the interactions of these drugs with DNA. In this study, fluorescent CdTe quantum dots (QDs, λem = 525 nm) were employed to investigate the interactions of platinum cytostatics (cisplatin, carboplatin, and oxaliplatin) with DNA fragment (500 bp, c = 25 μg/mL). Primarily, the fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/mL) had no significant influence on QDs fluorescence; however cisplatin and oxaliplatin quenched the fluorescence significantly (average decrease of 20%) at the same concentration. Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Best results were reached using oxaliplatin (9.4% quenching). Linear trend (R(2) = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125, and 0.063 mg/mL). Correlation with differential pulse voltammetric measurements provided linear trend (R(2) = 0.9511). As a conclusion, especially in the case of oxaliplatin-DNA adducts, the quenching was the most significant compared to cisplatin and nonquenching carboplatin.

Characterization of Oxaliplatin−DNA Adduct Formation in DNA and Differentiation of Cancer Cell Drug Sensitivity at Microdose Concentrations

(trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

Ultratrace voltammetric determination of DNA-bound platinum in patients after administration of oxaliplatin.

Oxaliplatin, a novel diaminocyclohexane-platinum complex, is used for the treatment of metastatic colorectal cancer. The amount of DNA-adduct formation of this drug in white blood cells of patients is determined after isolation of the DNA by density gradient centrifugation and a four-step solid phase extraction procedure. DNA is quantified by UV spectrometry, and platinum is determined after mineralization of the DNA sample by adsorptive stripping voltammetry (formazone method). It is possible to determine Pt-nucleotide ratios in clinical samples down to five Pt atoms in 10(8) nucleotides, and the dynamic range of the method covers three orders of magnitude. An absolute amount of 25 microg of DNA is sufficient for such measurements. With the method described, the time-dependent formation of oxaliplatin DNA adducts can be monitored in clinical studies, which may help us to understand inter-individual differences in the responses of patients to oxaliplatin-based therapy.

The Efficiency and Fidelity of Translesion Synthesis past Cisplatin and Oxaliplatin GpG Adducts by Human DNA Polymerase β

DNA polymerase beta (pol beta) is the only mammalian DNA polymerase identified to date that can catalyze extensive bypass of platinum-DNA adducts in vitro. Previous studies suggest that DNA synthesis by pol beta is distributive on primed single-stranded DNA and processive on gapped DNA. The data presented in this paper provide an analysis of translesion synthesis past cisplatin- and oxaliplatin-DNA adducts by pol beta functioning in both distributive and processive modes using primer extension and steady-state kinetic experiments. Translesion synthesis past Pt-DNA adducts was greater with gapped DNA templates than with single-stranded DNA templates. In the processive mode pol beta did not discriminate between cisplatin and oxaliplatin adducts, while in the distributive mode it displayed about 2-fold increased ability for translesion synthesis past oxaliplatin compared with cisplatin adducts. The differentiation between cisplatin and oxaliplatin adducts resulted from a K(m)-mediated increase in the efficiency of dCTP incorporation across from the 3'-G of oxaliplatin-GG adducts. Rates of misincorporation across platinated guanines determined by the steady-state kinetic assay were higher in reactions with primed single-stranded templates than with gapped DNA and a slight increase in the misincorporation of dTTP across from the 3'-G was found for oxaliplatin compared with cisplatin adducts.