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1
Master's Thesis
Anuar Makhmut
Department of Biological Sciences
Ulsan National Institute of Science and Technology 2021
Monitoring the repair of oxaliplatin-induced DNA- DNA crosslinks by ultra-performance liquid chromatography-selective ion monitoring (UPLC-
SIM) assay in human cells
[UCI]I804:31001-200000499983 [UCI]I804:31001-200000499983
2
Anuar Makhmut
Department of Biological Sciences
Ulsan National Institute of Science and Technology
Monitoring the repair of oxaliplatin-induced DNA- DNA crosslinks by ultra-performance liquid chromatography-selective ion monitoring (UPLC-
SIM) assay in human cells
3
A thesis/dissertation submitted to
Ulsan National Institute of Science and Technology in partial fulfillment of the
requirements for the degree of Master of Science
Anuar Makhmut
Orlando D. Schärer
Monitoring the repair of oxaliplatin-induced DNA- DNA crosslinks by ultra-performance liquid chromatography-selective ion monitoring (UPLC-
SIM) assay in human cells
06.10.2021 Approved by
Advisor
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Anuar Makhmut
This certifies that the thesis/dissertation of Anuar Makhmut is approved.
06.10.2021
___________________________
Advisor: Orlando D. Scharer
___________________________
Kyung Jae Myung
___________________________
Anton Gartner
Monitoring the repair of oxaliplatin-induced DNA- DNA crosslinks by ultra-performance liquid chromatography-selective ion monitoring (UPLC-
SIM) assay in human cells
5 Thesis abstract
Oxaliplatin (cis-[(1R, 2R)-cyclohexanediamine-N, N’] oxalate(2-)-O, O’) platinum; Eloxatine) is an anti-tumor drug used primarily for the treatment of colon and rectal cancer, as well as cancers that respond poorly to cisplatin treatment. Its anti-tumor activity is mainly attributed to the formation of platinum-DNA adducts that interfere with DNA replication, transcription, and chromatin remodeling, eventually inducing apoptosis and cell death. By binding to the N7 positions of the purine
nucleobases, oxaliplatin can form 1,2-intrastrand crosslinks between adjacent guanines (60-65%) or adenine and guanine (20-25%), 1,3-intrastrand crosslinks between two guanines separated by a pyrimidine nucleotide (5-10%), and interstrand crosslinks between guanines on opposing DNA strands. However, it is still not known which of the different DNA crosslinks are the most clinically relevant and how various DNA repair pathways contribute to the resistance to this therapy. Therefore, there is a need for methods to accurately quantitate levels of oxaliplatin-induced DNA crosslinks to correlate adduct level with the biological response, as well as using it as a diagnostic and predictive tool of oxaliplatin therapy outcomes in the clinic. Here, reported is the development of ultra- performance liquid chromatography-selective ion monitoring mass spectrometry (UPLC-SIM MS) assay for measuring different DNA-DNA crosslinks formed by oxaliplatin. Individual UPLC-SIM assays were developed for 1,2-GG, 1,2-AG, 1,3-GCG, and 1,3-GTG intrastrand crosslinks and G-oxp- G interstrand crosslinks. The methods were fully validated with high accuracy and precision, and the limits of detection for all intrastrand crosslinks were determined to be in the range of 12.5 fmol. The digestion of platinated DNA to representative analytes, solid-phase extraction, and high-performance liquid chromatography enrichment of the analytes was fully optimized using oxaliplatin-treated DNA oligomers and calf-thymus DNA. We observed a linear increase of oxaliplatin-induced crosslinks with increasing oxaliplatin treatment, confirming that the assays were reproducible and applicable to double-stranded substrates. The formation and repair of intrastrand crosslinks were then quantitated from oxaliplatin-treated NER-deficient patient cell lines and WT-XPA complemented controls, to test the hypothesis that NER-deficient cells will have a higher level of crosslinks due to reduced repair compared to wild type cells. As expected, in wild-type XPA cells the levels of oxaliplatin-induced DNA crosslinks increased up to 2 hours repair, reaching a maximum of 0.32 adducts per 106
nucleobases, followed by decreasing below the limit of quantitation after 24-hour repair. Conversely, in XPA deficient cells crosslink levels stayed consistent between 0.18 and 0.44 adducts per 106 nucleobases even after 24-hour repair time. In the future, this assay will be used to investigate the involvement of other repair pathways (TC-NER, BER, FA pathway, etc.) in removing platinum- induced DNA crosslinks and observing the difference in DNA crosslink formation and repair in platinum-sensitive and resistant cell lines.
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Contents
Ⅰ. List of figures ---8
Ⅱ. List of tables and schemes ---9
ⅡI. Technical terms and abbreviations ---10-11 IV. Introduction ---12-16 V. Experimental methods & materials ---16-21 VI. Results ---22-33 VII. Discussions ---33-36 VIII. Conclusion ---37 IX. References ---38-40 X. Acknowledgements---41
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List of figures
Figure 1: Oxaliplatin mechanism of action………...13
Figure 2: Types of DNA-DNA crosslinks formed by oxaliplatin……….13
Figure 3: Mechanisms of resistance of tumors to treatment……….15
Figure 4: OXP-d(GpX) authentic standards……….22
Figure 5: Representative UPLC-SIM chromatogram of the most abundant isotopes of doubly charged OXP-d(GpX) analytes………23
Figure 6. Representative fragmentation pattern of OXP-d(GG) analyte………..23
Figure 7. Retention profiles of OXP-d(GpX) on Waters HSS T3 column………...25
Figure 8. Monitoring two most abundant isotopes of OXP-d(GTG) and IS-OXP-d(GTG)………….26
Figure 9. Validation of the developed CP-d(GpX) assays………28
Figure 10. Digestion of 42mer oligo by PdeII………..29
Figure 11. Digestion of 42mer oligo by ExoI………...29
Figure 12. Digestion of 42mer oligo by ExoIII………....29
Figure 13. LC-MS confirmation of the 42mer 1,3-intrastrand crosslink digestion product after incubation with PdeII………30
Figure 14. Quantitation of OXP-d(GpX) in oxaliplatin-treated CTDNA………....32
Figure 15. Repair of OXP-d(GpX) in XPA wild type and deficient cells………....………33
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List of tables
Table 1: Observed m/z (+2) of each authentic standard and internal standard used for confirmation and quantitation………..20 Table 2. Table of observed OXP-d(GpX) authentic standard fragments from UPLC-PRM
experiments………....24 Table 3. Effect of pH on CP-d(GpX) sensitivity………...25 Table 4. Validation of the developed CP-d(GpX) assays……….27
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Technical terms and abbreviations
AAS Atomic absorption spectroscopy
ACN Acetonitrile
AMS Accelerator mass spectrometry
BER Base excision repair
CTDNA Calf thymus DNA
CV column volume
DMEM Dulbecco’s modified eagle’s medium
EDTA Ethylenediaminetetraacetic acid
ESI Electrospray ionization
FA Fanconi anemia
FBS Fetal bovine serum
FOLFOX Folinic acid, fluorouracil, oxaliplatin therapy
FPLC Fast protein liquid chromatography
GSH Glutathione
GSTP Glutathione transferase P
HESI Heated electrospray ionization
HPLC High-performance liquid chromatography
HLB Hydrophilic lipophilic balance
HR Homologous recombination
HRMS High-resolution mass spectrometry
HSS High strength silica
ICL Interstrand crosslink
ICP-MS Inductively coupled plasma mass spectrometry
LCMS Liquid chromatography-mass spectrometry
LOD Limit of detection
LOQ Limit of quantification
LRRC8 Leucine-rich repeat-containing protein 8
MeOH Methanol
MRP2 Multidrug resistance-associated protein 2
NER Nucleotide excision repair
NucP1 Nuclease P1
NucS1 Nuclease S1
OCT2 Organic cation transporter 2
OXP Oxaliplatin
PBS Phosphate buffered saline
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PDEII Phosphodiesterase II
PRM Parallel reaction monitoring
RCC Renal cell carcinoma
RP Reverse phase
SIM Selective ion monitoring
SPE Solid-phase extraction
TBE Tris/Borate/EDTA
TC-NER Transcription coupled NER
TLS Translesion synthesis
UV Ultraviolet
UPLC Ultra performance liquid chromatography
VRAC Volume-regulated anion channel
XPA Xeroderma pigmentosum complementation group A
XR-seq Excision repair sequencing
5-FU 5-fluorouracil
12 Introduction
Despite recent advances in targeted and immune therapies, platinum-based chemotherapies remain a primary method of treating a variety of solid tumors. The first platinum-based drug used in the clinic was cisplatin, which was first used to treat small cell lung cancer and is now mainly used in the treatment of testicular germ cell tumors, which are sensitive to cisplatin because of low NER activity1. Although cisplatin treatment has a high success rate for treating testicular and ovarian cancers, its usage is limited by serious side effects such as neurotoxicity, nephrotoxicity, and ototoxicity2, and it is not effective against certain type of cancers. Moreover, some cancer patients have tumors that are intrinsically resistant or acquire resistance during treatment. To alleviate these limitations while maintaining efficacy for tumor elimination, a lot of efforts have been made to synthesize new platinum drugs, but to this day, only 2 additional drugs have been approved medication for cancer treatment in humans: oxaliplatin and carboplatin.
Oxaliplatin, which was approved for clinical use in the EU in 1999 and in the USA in 20023, is a novel platinum-based antitumor drug, having less toxicity compared to cisplatin while being effective against gastrointestinal, colon, and rectal cancers where cisplatin shows minimal efficacy4. In the clinic, it is used in combination with 5-FU and folinic acid5, and its antitumor activity is mainly attributed to the formation of platinum-DNA adducts on the N7 position of guanines, which if not repaired, interfere DNA replication, transcription, chromatin remodeling and eventually causing apoptosis and cell death6.
Structurally, oxaliplatin is composed of a platinum atom bound to bidentate ligand 1,2-
diaminocyclohexane and an oxalate ligand susceptible to displacement. It is given by intravenous injection and enters the cell through organic cation transporter 2 (OCT2). Evidence suggest that oxalate ligand is replaced by water molecules7, 8, and as a result, several reactive species can be formed, including monoaquo and diaquo complexes, which can then react with nucleophilic positions on various biomolecules, such as amino groups of proteins, free amino acids, and N7 positions of DNA on guanine nucleobases, forming platinum-DNA adducts9. With DNA, oxaliplatin mainly forms intrastrand crosslinks between adjacent guanines or adjacent adenine and guanine, oxp-d(GG) and oxp-d(AG), intrastrand crosslinks between guanines separated by a pyrimidine nucleotide, oxp- d(GTG) and oxp-d(GCG), and interstrand crosslinks, G-oxp-G, formed by nucleotides in opposing strands of DNA and possibly being the most biologically relevant type of adduct10 (Figure 1).
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These crosslinks occur at very different levels: oxp-d(GG) and oxp-d(AG) account for 60-65% of all DNA adducts formed, 1,3-intrastrand crosslinks for 5-10%, interstrand crosslinks for 1-3%10.
Structurally they are discreet, exerting different distortion effects on the DNA helix. Importantly, it is not known how each type of crosslink contributes to the toxicity of oxaliplatin, which adducts are the most therapeutically relevant, and how various DNA repair pathways are involved in the removal of oxaliplatin-DNA adducts and how they contribute to the resistance to the therapy (Figure 2).
One of the biggest challenges in platinum-based cancer therapy is understanding mechanisms of intrinsic and acquired resistance of tumors to treatment. Mechanisms of resistance to therapy include increased level of DNA repair11, impaired apoptosis response12, increased damage response
pathways13, increased level of drug inactivation (GSH)14, and drug influx/efflux15. Knowledge of the clinical relevance of these mechanisms, however, is limited and incomplete.
Various DNA repair pathways are important for the removal of platinum-DNA lesions, and considerable efforts were dedicated to correlating variations in DNA repair capacity and DNA
Figure 2. Types of DNA-DNA crosslinks formed by oxaliplatin. The major DNA lesions are intrastrand crosslinks and interstrand crosslinks. The percentages represent the frequency of each type of DNA damage induced by oxaliplatin.
Figure 1. Oxaliplatin mechanism of action. Oxaliplatin enters the cell through OCT2, where it undergoes aquation which yields highly reactive species that can react with DNA, forming DNA-DNA crosslinks, that if not repaired, leads to apoptosis and cell death.
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damage response gene expression levels and polymorphisms to platinum drug responses16. In general, platinum-DNA lesions are helix-distorting lesions, and therefore removed by NER pathway to remove them. For example, it has been shown that the lesions formed by three platinum drugs, cisplatin, oxaliplatin, and satraplatin are repaired in vitro by the mammalian nucleotide excision repair pathway with similar kinetics17. However, the efficiency of repair of cisplatin-induced crosslinks is highly heterogeneous and significantly correlates with the states of chromatin folding and transcription18. Recently, genome-wide Damage-seq and XR-seq revealed that cisplatin-induced DNA damage and repair patterns are associated with several factors and that the damage formation is globally uniform and that the overall accumulated damage effect is not driven by not damage formation but repair efficiency19. The rate of excision repair also positively correlates with the gene expression, and repair is higher in the genes undergoing active transcription19.
Slyskova et al. have shown that by performing CRISPR-Cas9 screening to identify proteins that protect cells from oxaliplatin-induced DNA damage, that in addition to NER, HR, FA pathway, and TLS pathway, TC-NER and BER are also involved in protecting DNA against platinum drug-induced damage16. These pathways are crucial in removing oxaliplatin-induced crosslinks to ensure proper replication and transcription. Also, exposure to platinum drugs usually generates reactive oxygen species that generate oxidative DNA damage, which may explain the requirement of BER. This shows that the repair of platinum drug-induced DNA damage requires several repair pathways to restore functional DNA16. However, it is still inconclusive which repair pathways are crucial in determining cancer cell sensitivity and resistance.
Reduced drug accumulation was also shown to be a major feature of platinum drug-resistant cells and has been mainly attributed to defective drug import instead of increased efflux of the drug20. For cisplatin and carboplatin, it was found that accumulation occurs mainly through volume-regulated anion channels (VRACs) composed of LRRC8 heteromers21. Loss of subunits of LRCC8 was found to increase resistance to clinically relevant doses of cisplatin and carboplatin21. It was found that OCT2 is needed for intracellular accumulation of oxaliplatin and that its overexpression of it has a pronounced effect on the cytotoxicity of oxaliplatin22. Moreover, multidrug resistance-associated protein 2 (MRP2) can extrude unconjugated oxaliplatin from cells23. Another study found that MRP2 was the only one to be differentially expressed in the tumors of colorectal cancer patients who did or did not respond to FOLFOX chemotherapy, and over-expression of MRP2 decreased oxaliplatin accumulation and cytotoxicity but those deficits were reversed by inhibition of MRP2 with myricetin or siRNA knockdown24.
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Given that all potential resistance mechanisms influence the abundance of DNA-DNA crosslinks, and that platinum-DNA adducts have been considered useful biomarkers for monitoring oxaliplatin activity in cells, there is a need for an assay that will enable identification and quantification of each type of crosslink formed by oxaliplatin and correlation of adduct levels to the biological response.
Currently, there are several methods to assess levels of platinum drug-induced DNA crosslinks, including: using antibody probes against Pt-DNA damage25, AAS26, the COMET assay27, 32P postlabeling28, AMS10, and LC-ESI-MS29. Quantitation using antibody probes has the advantage in terms of simplicity in use and availability but has a disadvantage due to cross-reactivity and yield inconsistent results and are only made for detecting 1,2-intrastrand crosslinks. Neither the AAS nor the COMET assay can differentiate between the different types of platinum-DNA crosslinks, making them methods with limited availability. The 32P postlabeling method has the advantage over those methods by being able to distinguish different crosslink types and better sensitivity but requires the use of radioactivity and the procedure is labor-intensive. Accelerator mass spectrometry is sensitive enough to detect as low as 1 amol DNA adduct per µg of DNA and is the highest sensitivity method reported but requires the use of a highly specialized instrumentation that is not commonly available, making it impractical and labor-intensive.
For these reasons, LC-ESI-MS methods are superior in terms of user-friendliness, reproducibility, selectivity, sensitivity, and accuracy. Furthermore, recent developments in instrumentation and analytical separations have pushed limits of analytical techniques to quantify DNA adducts, allowing for the accurately assessment of levels of DNA adducts in various samples. An example of this methodology is a liquid chromatography-mass spectrometry method developed for the detection of DNA intrastrand crosslinks formed by oxaliplatin in wild-type and GSTP null mice29. However, in this method, only 1,2-intrastrand crosslinks were measured, overlooking the levels of 1,3-intrastrand crosslinks and interstrand crosslinks. Since 1,3-intrastrand crosslinks and interstrand crosslinks are substrates for nucleotide excision repair (NER) and Fanconi anemia pathway respectively,
quantitation of those crosslinks is important to understand the role of DNA repair in oxaliplatin toxicity and resistance.
Figure 3. Mechanisms of resistance of tumors to treatment. Cells can block oxaliplatin from reaching and damaging DNA by decreasing drug uptake, increasing drug efflux, increasing DNA damage repair and drug detoxification by covalent binding to glutathione.
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To address these issues, I developed is a comprehensive UPLC-HRMS-SIM assay to quantitate 1,2- and 1,3-intrastrand crosslinks in oxaliplatin-treated in vitro and cell culture samples. I was able to accurately quantitate the repair of oxaliplatin-induced intrastrand crosslinks in oxaliplatin-treated NER-deficient and isogenic control cells, demonstrating that the method is sensitive enough to detect crosslinks in vivo. In future studies, accurate quantitation of oxaliplatin-induced crosslinks will have the potential to correlate adduct levels and resistance and using it as a diagnostic and predictive tool of oxaliplatin therapy outcomes in the clinic.
Experimental Methods & Materials
Chemicals. Unless otherwise stated, all chemicals and enzymes were purchased from Merck & Co.
(Kenilworth, NJ). Isotopically labeled 15N5-labeled phosphoramidites were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). All reagents used for DNA oligonucleotide synthesis were purchased from Glen Research (Sterling, VA). Exonuclease I, exonuclease III, and quick calf intestinal alkaline phosphatase (Quick CIP) and nucleoside digestion mix were purchased from New England Biolabs (NEB, Ipswich, MA). DNase was purchased from Roche (Basel, Switzerland). 6 cc Oasis HLB cartridges were purchased from Waters Corporation (Milford, Massachusetts). Nucleoside digestion mix was purchased from New England Biolabs (Ipswich, Massachusetts). Illustra MicroSpin Columns were purchased from GE Healthcare (Chicago, Illinois).
Ultra-pure (UP) water was purchased from Biosesang (Seongnam, South Korea).
Preparation of OXP-d(GpX) analyte standards. Authentic standards of OXP-d(GpX) digestion products were synthesized for UPLC-SIM assay development and optimization. 5’-GG, 5’-AG dimers and 5’-GCG, and 5’-GTG dimers were prepared using standard DNA oligo synthesis protocols on a Mermade 4 automated DNA synthesizer. HPLC purification was performed using an Agilent 1260 Infinity HPLC coupled with an Agilent 1260 Infinity II photodiode array detector and a Phenomenex Clarity 5 m Oligo-RP (150 x 4.6 mm) column. A gradient of 100mM triethylamine acetate (TEAA, buffer A) and 100% methanol (buffer B) was operated at 1 mL/min starting at 2% B for 2 min, linearly increased to 9% B over 10 minutes, then 25% B over 10 minutes, then 80% B over 6 minutes, held constant at 80% B for 2 minutes, followed by a decrease to 2% B over 1 minute, and finally re- equilibrated at 2% B for 9 minutes. Under these conditions, AG dimer, GA dimer, GG dimer, GCG trimer, and GTG trimer eluted at 22.1, 21.2, 19.3, 21.4, and 22.8 min respectively.
HPLC-purified dimers and trimers were characterized by liquid chromatography-mass spectrometry (LC-MS) with a full-MS negative mode assay using a Q-Exactive Focus mass spectrometer coupled to a Dionex Ultimate 3000 UPLC system as follows; A Thermo Hypersil-Gold 1.9 μm C18 column (100 x 2.1 mm) was operated using a gradient of 15mM ammonium acetate, pH 7.0 (buffer A) and 100% Acetonitrile (buffer B) at 0.05 mL/min starting at 2% B for 2 min, linearly increased to 80% B over 16 minutes, held constant at 80% B for 2 minutes, followed by a decrease to 2% B over 2 minute, and finally re-equilibrated at 2% B for 12 minutes. MS settings were as follows; Scan range
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150 – 2000 m/z, electrospray voltage (3000 V), automatic gain control (AGC, 1e6), capillary
temperature 320 °C, HESI temperature 150 °C, Sheath gas, auxiliary gas, and sweep gas flow rate 35, 10, and 1 arbitrary unit respectively. ESI-MS (GG): m/z (-1) = 595.1400; ESI-MS (AG or GA): m/z (- 1) = 579.1444; ESI-MS (GCG): m/z (-1) = 884.1856; ESI-MS (GTG): m/z (-1) = 889.1853. Stock concentrations of each dimer and trimer were determined using UV absorbance measured on a NanoDrop 2000/2000c spectrophotometer using the following extinction coefficients: AG 260 = 25000, GA 260 = 25200, GG 260 = 21600, GCG 260 = 28200, and GTG 260 = 30300.
In an Eppendorf tube, dimer or trimer was incubated with 12 equivalents of regular oxaliplatin for 72 hours at 37°C protected from light. OXP-d(GpX) was purified by the HPLC method described above with OXP-d(AG), OXP-d(GG), OXP-d(GCG) and OXP-d(GTG) eluting at 17.4 (broad), 16.1, 19.8, and 21.9 minutes respectively. Each OXP-d(GpX) standard was characterized by a UPLC-parallel reaction monitoring (PRM) assay in positive mode as follows; A Waters HSS T3 1.8 μm C18 column (100 x 1.0 mm) was operated using a gradient of 15mM ammonium acetate, pH 7.0 (buffer A) and 100% methanol (buffer B) at 0.05 mL/min starting at 2% B for 2 min, linearly increased to 25% B over 8 minutes, followed by an increase to 50% B over 10 minutes, then an increase to 80% over 2 minutes, held constant at 80% for 2 minutes, followed by a decrease to 2% B over 2 minutes, and finally re-equilibrated at 2% B for 9 minutes. MS settings were as follows; Electrospray voltage (3000 V), capillary temperature (320 °C), full scan AGC (1e6), full scan resolution 70,000, HESI
temperature 150 °C, Sheath gas, auxiliary gas, and sweep gas flow rate 35, 10, and 1 arbitrary unit respectively, PRM AGC (5e4) and PRM resolution 35,000. Due to the natural occurrence of platinum isotopes, the three most abundant m/z were analyzed by UPLC-PRM as follows. ESI+-PRM OXP- d(GG): m/z (+2) = 452.11283, 452.61382, and 453.11421 from 12 – 14 min; ESI+-PRM OXP-d(AG):
m/z (+2) = 444.11543, 444.61646, and 445.11693 from 13.0 – 15 min; ESI+-PRM OXP-d(GCG): m/z (+2) = 596.63555, 597.13655, and 597.63727 from 12.5 – 14.5 min; ESI+-PRM OXP-d(GTG): m/z (+2) = 604.13544, 604.63659, and 605.13700 from 14 – 16 min.
Preparation of 15N5-labeled OXP-d(GpX) internal standards. Isotopically labeled internal standards of OXP-d(15N5-GG), OXP-d(15N5-AG), OXP-d(15N5-GCG), and OXP-d(15N5-GTG) were synthesized and characterized in the same way as the OXP-d(GpX) standards. In short, 15N5-labeled 5’-3’ GG, AG, GCG, and GTG dimers and trimers were prepared using a Mermade 4 automated DNA synthesizer. The coupling of the 15N5-labeled nucleotide was performed manually by adding 200 µL 67mM 2’-deoxyadenosine (15N5) or 15N5-2’-deoxyguanoside (15N5) phosphoramidite as the 5’-terminal guanines. Dimer/trimer HPLC purification, OXP-d(GpX) synthesis, and purification was performed exactly as described above for the authentic standards. Each 15N5-OXP-d(GpX) internal standard was characterized by the UPLC-parallel reaction monitoring (PRM) assay in positive mode described above for the authentic standards with the following changes; ESI+-PRM OXP-d(15N5-GG): m/z (+2)
= 454.60489, 455.10620 and 455.60656 from 12 – 14 min; ESI+-PRM OXP-d(15N5-AG): m/z (+2) = 446.60782, 447.10833 and 447.60916 from 13.0 - 15 min; ESI+-PRM OXP-d(15N5-GCG): m/z (+2) =
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599.12739, 599.62888 and 600.12960 from 12.5 – 14.5 min; ESI+-PRM OXP-d(15N5-GTG): m/z (+2)
= 606.62755, 607.12887 and 607.62941 from 14 - 16 min.
Synthesis of 42mer intrastrand crosslink substrate.
Activation of oxaliplatin was done by incubating 0.019 mmol (7.5 mg) oxaliplatin and 0.038 mmol of silver nitrate in 1 ml ultra-pure water overnight at 37°C protected from light. After incubation, the resulting silver chloride precipitate was removed using a 0.2mm nylon filter yielding a 21mM solution of activated oxaliplatin. To a labeled Eppendorf tube, 20 nmol of 42mer oligo was diluted to 600 μL at a final concentration of 10mM sodium perchlorate (NaClO4) and 5mM acetic acid and treated with 3 equivalents of aquated oxaliplatin for 1 hour at 37 °C protected from light. The sequence of the oligonucleotides was as follows; 5’-TCT TCT TCT TCT TCT TCT GGT TCT TCT TCT TCT TCT TCT TCT-3’ (42mer-GG XL), 5’-TCT TCT TCT TCT TCT TCT AGT TCT TCT TCT TCT TCT TCT TCT-3’ (42mer-AG XL), 5’-TCT TCT TCT TCT TCT TCT GTG TCT TCT TCT TCT TCT TCT TCT-3’ (42mer-GTG XL), and 5’-TCT TCT TCT TCT TCT TCT GCG TCT TCT TCT TCT TCT TCT TCT-3’ (42mer-GCG XL).
After incubation, excess oxaliplatin was removed using a 0.2 μm nylon filter. The 42mer 1,2-intra or 1,3-intrastrand crosslink oligo substrates were purified using an AKTA Pure FPLC as follows; A MonoQ 5/50 GL column was operated using a gradient of (A) 10mM NaOH and (B) 1M NaCl in 10mM NaOH at 2 mL/min starting at 10% B for 10 CV to equilibrate, then kept at 10% B for 5 CV after sample injection, followed by increasing to 30% B over 5 CV, then to 50% B over 40 CV, then to 100% B over 20 CV, held constant for an additional 5 CV and then decreased to 10% B to re- equilibrate for 10 CV. Under these conditions, the 42mer 1,2- and 1,3-intrastrand crosslink substrate eluted between 62 – 65 CV, and the unplatinated 42mer oligo was not observed. All collected substrate was dried using lyophilization. The resulting solid was resuspended in 1 mL of UP-water and desalted using centrifugal filtration (Merck, Amicon® Ultra 10 kDa filters) at 14,000 rcf for 10 minutes at 4 °C. The sample was washed with UP-water an additional three times to ensure buffer exchange. The product was confirmed by the negative mode full-scan LC-MS method described above.
Evaluation of digestion enzymes. A 100 pmol aliquot of a 42mer oligonucleotide containing a site- specific 1,2- or 1,3-intrastrand crosslinks were incubated in the presence of a single exonuclease as described in detail below. Digestion conditions, enzyme concentrations, and digestion times were optimized during analysis. The digestion reactions were quenched by adding an equal volume of 90%
formamide with bromophenol blue and then resolved on a 16cm x 20cm 20% urea gel with 1X TBE buffer at 300 volts for 2.5 hours. The resulting digestion products were visualized by staining with SYBR Gold and analyzed with an Amersham™ Typhoon™ biomolecular imager (GE Healthcare).
As an additional confirmation of digestion efficiency, the digestion of the 42mer oligonucleotides were repeated and the reaction products were HPLC purified using the OXP-d(GpX) method
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described above. Under these conditions, the desired 24mer or 21mer containing an oxaliplatin 1,2- or 1,3-intrastrand crosslink eluted as a broad peak between 25 – 28 min.
Platination and preparation of calf thymus DNA (CTDNA). A stock solution of CTDNA was resuspended in water at a concentration of 1 mg/mL. CTDNA (500 μg) was reacted with an increasing concentration of aquated oxaliplatin (50 nM, 100 nM, 250 nM, 500 nM, 1 µM, 2 µM, 5 µM, and 10 µM), brought to a final volume of 650 µL, and incubated at 37 °C for 24 hours protected from light.
After incubation, excess oxaliplatin was removed by centrifugal filtration (Merck, Amicon® Ultra 3 kDa filters) at 14,000 rcf for 10 minutes at 4 °C. The platinated CTDNA was further washed with an equal volume of UP-water three additional times, followed by recovering the CTDNA following the manufacturer’s protocol. The concentration of the platinated CTDNA concentration was measured using the double-strand DNA (dsDNA) settings on a microvolume UV spectrophotometer (Thermo Scientific™ NanoDrop 2000). Purified platinated CTDNA solutions were stored at -20 °C.
Digestion and sample enrichment of CTDNA for in-vitro quantitation of OXP-d(GpX). Aliquots of platinated CTDNA (50 g to 100 g) were spiked with 2 pmol of OXP-d(GpX) IS, and the samples enzymatically digested to OXP-d(GpX) analytes using the following procedures. While optimizing digestion methods, technical replicates (N = 2 or 3) were analyzed to confirm reproducibility. After digestion optimization for each OXP-d(GpX), the CTDNA platination described above was repeated to obtain experimental replicates (N = 3) of OXP-d(GpX) quantitation.
For the digestion, a 25 µg aliquot of OXP-treated CTDNA was diluted to 100 µL of 1x nucleoside digestion mix reaction buffer (NEB, Ipswich, MA) at pH 5.4 and treated with 3.3 µL NEB nucleoside digestion mix (1 µL per 7.5 g CTDNA) for 1 hour (OXP-d(GTG) and OXP-d(GCG)) or 4 hours (OXP-d(AG) and OXP-d(GG)) at 37 °C. Immediately after digestion, the reaction was quenched by the addition of 10 mM EDTA and 5% methanol and the samples were processed as described below.
Enzyme removal. The digestion enzymes of each reaction were removed by purifying crude mixture using Oasis HLB disposable SPE columns. The columns were placed in the vacuum manifold, and the vacuum was set to 5 mmHg. Columns were washed with 1 ml methanol before equilibration with 1 ml ultra-pure water. The crude mixture was allowed to pass through the column, washed with 1 ml of 5%
methanol, and eluted with 1 ml 100% methanol. All collected solutions were concentrated to dryness by vacuum centrifugation.
HPLC enrichment. OXP-d(GpX) was enriched by HPLC purification using an Agilent 1260 Infinity HPLC coupled with an Agilent 1260 Infinity II photodiode array detector and a Phenomenex Clarity 5 m Oligo-RP (150 x 4.6 mm) column following the methodology described above for OXP-d(GpX) authentic standards. During analysis, samples were collected in 1-minute fractions from 11.0 – 22.0 minutes were collected by the automated fraction collector. Fractions from 15.0 – 17.0 minutes (OXP- d(GG)), 17.0 – 19.0 minutes (OXP-d(AG)), 19.0 - 21.0 minutes OXP-d(GCG)), and 21 – 22 minutes (OXP-d(GTG)) were pooled together and concentrated by vacuum centrifugation and lyophilization.
The concentrated samples were resuspended in 20 µL LC-MS water for UPLC-SIM analysis.
20
Ultra-performance liquid chromatography-single ion monitoring (UPLC-SIM) of OXP-d(GpX).
Each OXP-d(GpX) analyte was analyzed using UPLC-SIM assays in positive mode as follows; A Waters HSS T3 1.9 m C18 column (100 x 1.0 mm) was operated using a gradient of 15mM
ammonium acetate, pH 7.0 (buffer A), and 100% methanol (buffer B) at 0.05 mL/min starting at 2%
B for 2 min, linearly increased to 25% B over 8 minutes, followed by an increase to 50% B over 10 minutes, then an increase to 80% over 2 minutes, held constant at 80% for 2 minutes, followed by a decrease to 2% B over 2 minutes, and finally re-equilibrated at 2% B for 9 minutes. MS settings were as follows; Electrospray voltage (3000 V), capillary temperature 320 °C, HESI temperature 150 °C, Sheath gas, auxiliary gas, and sweep gas flow rate 35, 10, and 1 arbitrary unit respectively. Due to the natural occurrence of platinum isotopes, the two most abundant m/z were detected for both the analyte and internal standard by UPLC-SIM, respectively, as follows. ESI+-SIM OXP-d(GG): m/z (+2) = 452.61382 and 453.11421; 455.10620 and 455.60656 from 12 – 14 min; ESI+-SIM OXP-d(AG): m/z (+2) = 444.61646 and 445.11693; 446.60782, and 447.10833 from 13 – 15 min; ESI+-SIM OXP- d(GCG): m/z (+2) = 597.13655 and 597.63727; 599.62888 and 600.12960 from 14 – 16 min; ESI+- SIM OXP-d(GTG): m/z (+2) = 604.63659 and 605.13760; 607.12887 and 607.62941 from 12.5 – 14.5 min. To avoid any potential overlap of analyte and internal standard isotope signals, the third isotope signal (196Pt) was used for quantitation and the more abundant second isotope signal (195Pt) was used for confirmation (Table 1). OXP-d(GpX) quantitation was performed by dividing the area under the analyte signal by the internal standard signal and multiplying by the amount of internal standard spiked into the sample.
OXP-d(GpX) UPLC-SIM assay validation. Aliquots of 25 µg CTDNA were spiked with 2 pmol OXP-d(GG), OXP-d(AG), OXP-d(GCG), or OXP-d(GTG) IS and the following amounts of authentic standards (N = 3): 500, 250, 100, 40, or 20 fmol. For 1,2- and 1,3-intrastrand crosslinks, CTDNA was
Structure Analyte confirmation
Analyte quantitation
IS
confirmation IS quantitation
OXP-d(GG) 452.6141 453.1139 455.1070 455.6072
OXP-d(AG) 444.6175 445.1176 447.1096 447.6099
OXP-d(GCG) 597.1383 597.6387 599.6302 600.1304
OXP-d(GTG) 604.6381 604.1386 607.1297 607.6299
Table 1. Observed m/z (+2) of each authentic standard and internal standard used for confirmation and quantitation.
21
digested by the enzyme digestion method as described above, followed by processing and enrichment by Oasis HLB cartridges and HPLC purification. Collected fractions were analyzed by UPLC-SIM as described above, and the observed ratio of analyte to internal standard was plotted against the
expected ratios. The accuracy of each point was calculated by subtracting the mean ratio by the expected ratio, followed by dividing by the expected ratio. The precision of each point was calculated by dividing the standard deviation by the mean ratio. The limit of detection of each assay was
determined as a signal-to-noise ratio of 3:1, and the limit of quantitation was determined as a signal- to-noise ratio of 10:1 from the validation results.
Oxaliplatin treatment of cell culture. Immortalized XPA-deficient XP2OS cells derived from a Xeroderma pigmentosum patient cells and cells complemented with WT protein by lentiviral transfection were seeded (1.0 x 106 cells) overnight in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO2. Cells were cultured and allowed to grow to 70% confluency before treatment.
A stock solution of 2.5 mM oxaliplatin was prepared in 154 mM NaCl solution and vortexed for 30 minutes to ensure everything was dissolved. The cell media was removed, cells were washed twice with phosphate-buffered saline, and cell medium containing 50 µM oxaliplatin was added. After 2 hours of incubation, the medium was removed, and cells were washed three times with PBS. Fresh cell medium was added, and the cells were incubated for an additional 1, 2, 6, or 24 hours to allow repair of OXP-induced DNA crosslinking. After the appropriate repair time, cells were trypsinized and collected for future processing. For the 0-hour time point, cells were harvested immediately after the two-hour incubation with 50 µM oxaliplatin.
DNA was extracted by first lysing the cells in Qiagen Cell Lysis Solution (1 mL per 1.0 x 106 cells).
Each solution was supplemented with 8 U proteinase K and gently inverted overnight at room temperature to improve cell lysis and digest DNases. After cell lysis, the proteinase K was
precipitated by adding 250 µL Qiagen Protein Precipitation Solution and vortexing for 30 seconds.
The precipitated proteins were pelleted by centrifugation at 4000 rpm for 10 minutes at 4 °C. The supernatant was decanted to labeled Eppendorf tubes and incubated with 70 U RNase at 37 °C for 5 hours. The RNase was precipitated and removed as described above, and DNA was precipitated by adding an equal volume of 100% ethanol. Extracted genomic DNA was digested and processed using the appropriate methodology described above.
22 Results
Synthesis and characterization of OXP-d(GpX) standards and internal standards.
Authentic standards and 15N5-labeled internal standards of OXP-d(GG), OXP-d(AG), OXP-d(GCG), and OXP-d(GTG) were synthesized as described in the experimental section using the appropriate dimer or trimer as a starting material and unactivated oxaliplatin. Each OXP-d(GpX) was purified using HPLC and characterized by UV spectroscopy, UPLC-SIM, and UPLC-PRM. Confirmation of the 15N5-labeled internal standard purity was achieved by the UPLC-SIM analysis as described in the experimental section (Figure 4).
Analysis of OXP-d(GpX) authentic standard by UPLC-SIM in positive mode revealed a cluster of peaks with +2 ionization states because naturally occurring platinum exists as a mixture of six non- radioactive isotopes (Figure 5). The isotope distribution served as an additional confirmation during UPLC-SIM development. The isotope of greatest abundance (195Pt, 33.8%) gives rise to the base peaks at 452, 444, 597 and 604 for oxp-d(GG), oxp-d(AG), oxp-d(GCG) and oxp-d(GTG) respectively.
OXP-d(GpX) was further characterized by UPLC-PRM fragmentation on the three most abundant isotopes for each authentic standard (Figure 6, Table 2). For all of OXP-d(GpX), the major fragments were breakage of the glycosidic bond and loss of one guanine to yield a single guanine moiety
crosslinked to platinum. For example, the most abundant isotope of the OXP-d(GTG) analyte yielded fragments of m/z (+1) = 459.15.12, m/z (+1) = 610.1724, and m/z (+1) = 152.0569, which
corresponded to loss of the glycosidic bond and guanine and thymine moieties, loss of glycosidic bond and thymine moiety, and 5’ guanine crosslinked to platinum, respectively (Table 2).
Figure 4. OXP-d(GpX) authentic standards. Each OXP-d(GpX) authentic standard has unique mass-to-charge (m/z) ratio which can be identified by mass spectrometer and represents simplest unit of each crosslink type.
23
Figure 6. Representative fragmentation pattern of OXP-d(GG) analyte. For OXP-d(GG), the major fragments after PRM experiments were [Gua-Pt(NH3)]+ and [Gua-Pt(NH3)-Gua]+.
Figure 5. Representative UPLC-SIM chromatogram of the most abundant isotopes of doubly charged OXP-d(GpX) analytes. Naturally occurring platinum exists as a mix of 6 non-radioactive isotopes. During analysis, he second most abundant isotope (195Pt) was used for confirmation, and the third most abundant isotope (196Pt) was used for quantitation.
24
UPLC-SIM assay development. Using the synthesized and characterized authentic standards and
15N-labeled internal standards, the conditions for UPLC-HRAM-MS were investigated. A C18 column (100 x 1.0 mm) was used to determine the retention profiles of OXP-d(GG), OXP-d(AG), OXP-d(GCG), and OXP-d(GTG) to 12.8, 14.06, 13.53 and 14.9 min respectively (Figure 7). To increase the sensitivity of detection, we hypothesized that lowering the pH of 15mM ammonium acetate buffer from 7.0 to 6.0 and 4.5 will increase the fraction of doubly charged species. However, when solutions of OXP-d(GpX) standards and internal standards were analyzed in low pH buffer conditions, the sensitivity decreased significantly, indicating that pH 7.0 was the optimal condition for detecting OXP-d(GpX) (Table 3).
Substrate Parent mass (m/z)
Observed fragment 1
(m/z)
Observed fragment 2
(m/z)
Observed fragment 3
(m/z)
OXP-d(GG)
452.1136 458.1195 305.0880 609.1686
452.6148 459.1217 305.5891 610.1710
453.1139 460.1222 306.0893 611.1767
OXP-d(AG)
412.0818 297.0907 458.1195 593.1738
412.5827 297.5917 459.1221 594.1768
413.0830 298.0902 460.1229
OXP-d(GCG)
556.6048 305.0885 458.1200 609.1690
557.1057 305.5896 459.1219 610.1713
557.6059 306.0892 460.1226 611.1722
OXP-d(GTG)
564.1047 305.0541 458.1205 609.1705
564.6060 305.5896 459.1221 610.1724
565.1100 306.0568 460.1225 611.1764
Table 2. Table of observed OXP-d(GpX) authentic standard fragments from UPLC-PRM experiments.
25
Figure 7. Retention profiles of OXP-d(GpX) on Waters HSS T3 column. Representative UPLC- SIM chromatogram of each OXP-d(GpX) that has unique retention time and thus can be separated and identified during liquid chromatography purification step of UPLC-SIM.
Analyte pH 7.0 (Analyte area)
pH 5.5 (Analyte area)
pH 4.0
(Analyte area) Comment
OXP-d(GG) 4631912 4177552 3899131 Sensitivity
decreased 15.8%
OXP-d(AG) 5133110 4831258 1001106 Sensitivity
decreased 80.4%
OXP-d(GCG) 8981443 7265953 5935108 Sensitivity
decreased 33.9%
OXP-d(GTG) 7804331 6455336 5198768 Sensitivity
decreased 33.3%
Table 3. Effect of pH on CP-d(GpX) sensitivity.
26
For additional confidence in OXP-d(GpX) detection, we chose to monitor the two most abundant isotopes. During analysis, the overlap of analyte and internal signals is possible. To avoid this, the second most abundant isotope (195Pt) was used for confirmation, and the third most abundant isotope (196Pt) was used for quantitation (Figure 8). Selective ion monitoring assay provided good overall selectivity and sensitivity to detect and differentiate each of the digested OXP-d(GpX) adducts.
The developed UPLC-SIM assays were validated by processing and analyzing untreated CTDNA (50 μg, N=3) with the addition of an increasing amount of authentic standard (50fmol - 1 pmol) and 2 pmol of internal standard as described in the experimental section. The obtained ratio of analyte to internal standard was plotted against the expected analyte to internal standard ratio to confirm linearity (r2 = 0.99) and calculate accuracy and precision for all OXP-d(GpX) adducts. The limit of quantitation (LOQ) was set to be a signal-to-noise ratio >10, and the limit of detection (LOD) was set to be a signal-to-noise ratio >3. From the validation experiments, the LOQs for all OXP-d(GpX) was 12.5 fmol. The individual accuracy and precision calculations for each point of the validation curves are provided in table 4 and figure 9.
Figure 8. Monitoring two most abundant isotopes of OXP-d(GTG) and IS-OXP-d(GTG).
Representative UPLC-SIM chromatogram of OXP-d(GTG) from 1µM oxaliplatin-treated ctDNA.
Panels 1, 2 are isotopes of OXP-d(GTG) and panels 3, 4 are the analogous isotopes of OXP-d(GTG).
27 Substrate Ratio
(Std:IS) Accuracy Precision Linearity LOQ LOD
OXP-d(AG)
0.025:1 61.17% 34.01%
y = 1.1321x – 0.0125 r2 = 0.9949
25 fmol 12.5 fmol
0.05:1 88.24% 6.80%
0.1:1 82.68% 8.07%
0.2:1 119.47% 11.76%
0.5:1 109.45% 3.50%
OXP-d(GG)
0.02:1 139.6% 42.36%
y = 1.0881x – 0.0143 r2 = 0.9948
25 fmol 12.5 fmol
0.04:1 93.7% 7.92%
0.08:1 104.5% 13.03%
0.2:1 97.8% 10.57%
0.4:1 95.3% 5.86
OXP- d(GCG)
0.025:1 42.79% 34.47%
y = 1.0425x - 0.0058 r2 = 0.9620
25 fmol 12.5 fmol
0.05:1 42.41% 24.14%
0.1:1 84.95% 17.10%
0.2:1 136.80% 7.36%
0.5:1 98.56% 17.14%
OXP- d(GTG)
0.025:1 91.6% 30.0%
y = 0.9591x + 0.0027 r2 = 0.9877
25 fmol 12.5 fmol
0.05:1 79.19% 13.62%
0.1:1 88.04% 9.85%
0.2:1 115.5% 3.01%
0.5:1 94.3% 5.75%
Table 4. Validation of the developed CP-d(GpX) assays.
28
Digestion of platinated DNA oligomers. The goal of this experiment was to show that the UPLC- SIM assays work on simple substrates such as platinated DNA oligomers containing a site-specific oxaliplatin crosslink. It was critical to identify the enzymes that are capable of digesting platinated DNA oligomers up to the crosslink without overdigesting the internal phosphodiester bond. For this, we first incubated a known amount of 42mer oligo containing a site-specific oxaliplatin crosslink with one or two exonucleases. After incubation, the digestion reaction mixture was resolved by 20% urea gel and digestion products visualized by SYBR gold staining. To maximize digestion efficiencies and avoid overdigestion of OXP-d(GpX), enzyme concentration and incubation times were optimized.
Incubation of 40 mU of the PDEII, which has 5’ - 3’ exonuclease activity for 4 - 24 hours with 100pmol 1,2- or 1,3-intrastrand crosslink oligomers successfully digested them up to the OXP- d(GpX) and showed no evidence of overdigestion (Figure 10).
Figure 9. Validation of the developed CP-d(GpX) assays. Untreated CTDNA was spiked with a known amount of authentic standard and IS, followed by digestion by NEB mix. The analyte: IS ratio was plotted against the theoretical ratio to establish linearity of assay.
29
Figure 10. Digestion of 42mer oligo by PdeII. Representative 20% urea gel of 42mer XL oligo digestion by PdeII to show that it can digest up to 1,2- and 1,3-intrastrand crosslinks.
Figure 11. Digestion of 42mer oligo by ExoI. Representative 20% urea gel of 100pmol 42mer XL oligo digestion by PdeII to show that it can digest up to 1,3-intrastrand crosslinks, but overdigests 1,2-intrastrand crosslink.
Figure 12. Digestion of 42mer oligo by ExoIII. Representative 20% urea gel of 100pmol 42mer XL oligo digestion by 3’-5’ exonuclease ExoIII, which is more active on dsDNA substrates and does not digest 1,2- and 1,3-intrastrand crosslinks under every condition investigated.
30
Increasing enzyme concentration of PDEII up to 80 mU did not result in observable overdigestion of the platinated oligomers. Digestion of 1,3-intrastrand crosslink oligomers with exonuclease I, (a 3’ - 5’ exonuclease), also successfully digested up to the OXP-d(GpX). However, in this case, increasing concentration, led to overdigestion of the OXP-d(GpX) as the band corresponding to the desired product, as indicated by a decreased intensity of the 24mer product band. The 1,2-intrastrand crosslink was overdigested, despite adjusting enzyme amount or the digestion time (Figure 11). We also attempted digestion with exonuclease III (a 3’ – 5’ exonuclease). Consistent with its higher activity on dsDNA substrates, but no noticeable digestion was observed based on the gel analysis (Figure 12).
The digestion products were purified by HPLC and analyzed by full-scan LC-MS to support the digestion optimization results described above.
The exact mass of the 24mer containing a crosslink at the 5’ end (1,3-GTG m/z = 7481.2236) was observed as the major product for the PDE II (Figure 13). Overall, it was confirmed that PDE II and Exo I were appropriate exonucleases that digest platinated DNA oligomer up to OXP-d(GpX), and when used in combination or with endonucleases, could potentially digest platinated DNA to yield OXP-d(GpX).
Digestion optimization of 1,2- and 1,3-intrastrand crosslinks on dsDNA substrates. Having shown that the developed UPLC-SIM methods are successful in enriching OXP-d(GpX) from simple substrates such as platinated DNA oligomers, we tested this approach on more complicated substrates, oxaliplatin-treated calf-thymus DNA (CTDNA). It resembles genomic DNA in size, being a double- stranded template DNA (dsDNA) but it does not contain any chromatin-associated proteins. It was used as an in-vitro substrate to optimize DNA digestion conditions due to its similarity to platinated genomic DNA.
We treated CTDNA with an increasing concentration of activated oxaliplatin (50 nM – 10 µM) and digested it using NEB nucleotide digestion mix (New England Biolabs, Ipswich, MA), which is an
Figure 13. LC-MS confirmation of the 42mer 1,3-intrastrand crosslink digestion product after incubation with PdeII. The digestion product was analyzed by full-scan MS to confirm that PdeII digested DNA up to the crosslink.
31
optimized mixture of enzymes that generates single nucleosides from DNA or RNA. After the addition of isotopically-labeled internal standards, digestion, and enrichment, OXP-d(GpX) were measured using the developed UPLC-SIM assays.
Earlier studies on quantitating oxaliplatin-induced DNA crosslinks used DNAse I, shrimp alkaline phosphatase, and Nuc P1 to digest oxaliplatin-treated DNA to enrich OXP-d(GpX) 29. Similarly, Hah et al utilized a combination of DNaseI and NucS1 to quantitate all possible oxaliplatin-induced DNA crosslinks10. I found that Nuc P1, although has been shown to successfully enrich 1,2-intrastrand crosslink, overdigested 1,3-intrastrand crosslinks, preventing accurate quantification of these adducts in the sample. A similar effect was observed with Nuc S1, which has long digestion times as an additional inconvenience. The commercially available NEB mix has been utilized to quantitate structurally analogous DNA crosslinks and was chosen as the main digestion method to enrich OXP- d(GpX) from various samples as it has shown to not show overdigestion, relatively short digestion time, and convenient enrichment of analytes after digestion.
After several digestion optimizations, I found that following digestion by NEB mix, OXP-d(GG) was detected at oxaliplatin treatments as low as 50 nM with 4.62 ± 0.23 OXP-d(GG) per 106 nucleotides (N = 3). Increasing oxaliplatin treatment of 100, 250, 500 yielded a linear signal increase of 8.01 ± 0.81, 18.20 ± 1.66, and 28.47 ± 0.54 OXP-d(GG) per 106 nucleotides (N = 3) respectively.
Interestingly, OXP-d(AG) was below LOQ when attempting to enrich with a similar linearly increasing dose-dependence set of oxaliplatin. It was detected, however, in 1, 2, 5, and 10 μM oxaliplatin dose-treated CTDNA and this also resulted in a linear increase of 4.42 ± 2.34, 10.05 ± 1.17, 20.96 ± 0.83, and 46.59 ± 3.47 OXP-d(AG) per 106 nucleotides (N = 3) respectively (Figure 14). This result does not coincide with the previous results and suggests that oxp-d(AG) does not represent 25% of all crosslinks formed by oxaliplatin under our conditions.
1,3-intrastrand crosslinks – OXP-d(GCG) and OXP-d(GTG) - represent less abundant crosslinks formed by oxaliplatin, OXP-d(GCG), and OXP-d(GTG) comprising 5-10% of all adducts formed.
Therefore, for the similar dose dependence experiment, CTDNA was treated at a higher dose to allow the formation of 1,3-intrastrand crosslinks at levels above LOQ. Oxaliplatin treatment at 1, 2, 5, 10 μM concentration resulted in 2.40 ± 0.04, 5.46 ± 0.28, 15.30 ± 0.59 and 27.45 ± 0.06 OXP-d(GTG) per 106 nucleotides (N = 3) respectively. Similarly, the levels of OXP-d(GCG) at 1, 2, 5, 10 μM concentration were 30.80 ± 13.56, 58.94 ± 18.55, 187.15 ± 63.64 and 292.41 ± 29.31 adducts per 106 nucleotides (N = 3) respectively.
32
Interestingly, when analyzing 1,3-intrastrand crosslinks, a shift in the retention time was observed, specifically, the OXP-d(GTG) and OXP-d(GCG) analyte peaks eluted earlier than the internal standard peaks (specify the retention time). When looking at the raw data, the shifted analyte peaks had the desired isotope distribution pattern and correct m/z ratios within 5 ppm. The unexpected peaks were possibly the products of rotamers. Therefore, we heated the samples to 80 ˚C for 10 minutes before UPLC-SIM analysis, but this did not result in a single peak with the same retention time as the internal standard. We hypothesize that the shift in the retention time of analyte peaks suggests that the observed peaks are the isomers of the desired OXP-d(GCG) and OXP-d(GTG). However, because of the identical MS analysis profile, they can only result from 1,3-intrastrand crosslinks.
OXP-d(GpX) quantitation in OXP-treated cell lines.
To demonstrate the application of the developed UPLC-HRAM-SIM assays to monitor the repair of oxp-d(GpX) in cells, we quantified OXP-d(GpX) repair over time in oxaliplatin-treated nucleotide excision repair-deficient XPA-/- cells and their WT-XPA complemented controls. Both cell lines were treated with 10 µM OXP for 2 hours, then incubated for 0-, 1-, 2-, 6-, or 24-hours for repair, and the adduct levels were quantified in each sample. For quantification of 1,2-intrastrand crosslinks, the DNA was extracted from a single 10cm dish and digested by NEB mix for 4 hours. After digestion, the analytes were enriched by Oasis HLB cartridges and HPLC purifcation, and analysis was done by developed UPLC-HRAM-SIM assays as described above.
Figure 14. Quantitation of OXP-d(GpX) in oxaliplatin-treated CTDNA. Aliquots of platinated CTDNA were spiked with 2 pmol of IS and processed as described above. Adduct levels are expressed as OXP-d(GpX) per 106 nucleotides.
y = 0.0529x + 2.9331 R² = 0.9829
0 5 10 15 20 25 30 35
0 100 200 300 400 500 600
adducts per 106nucleotides
oxaliplatin [nM]
oxp-d(GG) dose dependence
y = 4.6149x - 0.262 R² = 0.9951
0 10 20 30 40 50
0 2 4 6 8 10 12
adducts per 106nucleotides
oxaliplatin [μM) oxp-d(AG) dose dependence
y = 2.7879x + 0.1021 R² = 0.9944 0
5 10 15 20 25 30
0 2 4 6 8 10 12
adduct per 106nucleotides
oxaliplatin [μM ] oxp-d(GTG) dose depencdence
y = 29.524x + 9.4733 R² = 0.9724 500
100 150 200 250 300 350
0 2 4 6 8 10 12
adducts per 106nucleotides
oxaliplatin {μM]
oxp-d(GCG) dose dependence
33
In oxaliplatin-treated XPA wild-type cells, the OXP-d(GG) levels were 0.09 adducts per 106 nucleotides at 0-hour repair, increased to 0.16 adducts per 106 nucleotides after 2-hour repair, decreased to 0.14 adducts per 106 nucleotides after 6-hour repair, and no OXP-d(GG) was detected after 24-hour repair, indicating that the levels were below the limit of detection. Conversely, the levels of OXP-d(AG) were below the limit of detection in all samples and could not be quantitated in these samples. In oxaliplatin-treated XPA deficient cells, the OXP-d(GG) levels were 0.30 adducts per 106 nucleotides at 0-hour repair, remained relatively same after 2-hour repair, increased to 0.43 adducts per 106 nucleotides after 6-hour repair and then decreased to 0.18 adducts per 106 nucleotides after 24- hour repair. Although repair was observed in both groups, the trend is more evident in wild type cells and the levels of oxp-d(GG) are higher in deficient cells, indicating less efficient repair. Overall, our developed assay was able to detect OXP-d(GG) repair in XPA wild-type cells. Although the repair trend was unclear in XPA deficient cells, the OXP-d(GG) was still detected after 24-hour repair, indicating that the repair is not going efficiently.
Discussion
Oxaliplatin is an FDA-approved platinum-based cancer drug used primarily for treating
gastrointestinal and colorectal cancers. Despite its efficiency, oxaliplatin therapy is often accompanied by severe side effects such as chemotherapy-induced peripheral neuropathy, ototoxicity, as well as intrinsic or acquired resistance. Side effects and resistance are shaped by several factors, such as cellular import and efflux of the drug, drug metabolism and interaction, and DNA damage repair and tolerance. The knowledge of the clinical relevance of these mechanisms, however, is limited.
Additionally, there is currently no readily available and reliable approach that enables accurate quantification of oxaliplatin-induced DNA adducts and correlating them with the resistance to oxaliplatin therapy.
Figure 15. Repair of OXP-d(GpX) in XPA wild type and deficient cells. Cells were treated with 10 µM OXP for 2 hours, then incubated for 0-, 1-, 2-, 6-, or 24-hours for repair, and the adduct levels were quantified in each sample using the developed UPLC-SIM assay.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0h 1h 2h 6h 24h
Adducts per 106nucleotides
Repair time (hour)
oxp-d(GG) repair
XPA wild type XPA deficient
34
To understand how DNA repair pathways contribute to the resistance to therapy, we developed a comprehensive UPLC-SIM assay to measure intrastrand crosslinks formed by oxaliplatin. 1,2-GG intrastrand crosslinks, in agreement with previously developed mass spectrometry-based methods, was the most abundant crosslink formed by oxaliplatin. Similarly, 1,3-GTG and 1,3-GCG intrastrand crosslinks were less abundant and only consistently detected in cells treated with higher doses of oxaliplatin. In contrast, 1,2-AG intrastrand crosslink levels were not in agreement with previous reports. I was unable to detect cells and samples where 1,3-GTG and 1,3-GCG intrastrand crosslinks were detected, suggesting that it does not represent 25% of the total DNA adducts that were reported previously. The methods developed in this thesis, were fully validated and the limits of detection and limits of quantification were determined to be 25fmol and 12.5fmol, respectively. Using NEB mix for digestion and an optimized enrichment procedure, we quantified the levels of each intrastrand
crosslinks in oxaliplatin-treated CTDNA samples. For 1,2-GG intrastrand crosslink, we observed a linear dependence between oxaliplatin dose and crosslink levels was observed from a low dose set (50nM-500nM). For 1,2-AG, 1,3-GCG, and 1,3-GTG intrastrand crosslinks the linear relationship was established with a higher concentration (1μM - 10μM) since these crosslinks are less in abundance.
For the in vitro optimization experiments, we incubated the CTDNA with activated oxaliplatin in contrast to intact oxaliplatin used in the previous reports by the Farmer29 and Henderson10 laboratories respectively. Activated oxaliplatin may react with DNA more readily and this might affect the overall abundance of different types of crosslinks induced by oxaliplatin. With activated oxaliplatin, the levels of 1,2-GG and 1,2-AG intrastrand crosslinks were almost the same, while with unmodified oxaliplatin, Farmer observed 3.1±0.4 1,2-GG intrastrand crosslinks for every 1,2-AG intrastrand crosslink when analyzed dose-dependence set of 3.25nM to 16.25μM29. The kinetic studies from the Henderson group revealed that dG or dA mono adducts increase and then decrease 24-hour post- treatment, suggesting that monoadducts are intermediates during the formation of intrastrand crosslinks10. The abundance of 1,2-intrastrand crosslinks did not change significantly, and 1,3- intrastrand levels increased as the incubation time increased, the peak abundance reaching 36%, 24 hours post-incubation10. This may indicate higher thermodynamic stability 1,3-intrastrand crosslinks in comparison to the faster formation of 1,2-intrastrand crosslinks30. In addition to drug type and concentrations, differences in the method including buffer composition, enzymatic digestion, enrichment of digestion products may affect the overall abundance of each type of crosslink. In our methods, we used NEB mix to digest DNA, which despite showing signs of overdigestion at long incubation times, was shown to optimally enrich 1,3-intrastrand crosslinks after optimization. A key step in digestion optimization that aided in preventing overdigestion of analyte was the addition of EDTA, which is a chelating agent that removes metal ion cofactors from enzymes leading to their inactivation.
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DNA damage repair pathways have an important role in determining sensitivity and resistance of cancers to platinum drug-based chemotherapy; therefore, many efforts have been made to correlate DNA damage repair efficiency and changes in DDR gene expression to responses of tumors to platinum drugs. Since oxaliplatin induces various DNA crosslinks such as intra- and interstrand crosslinks, repair of those crosslinks requires the involvement of several repair pathways. Lans found that in addition to the FA pathway, HR, TLS, and NER, TC-NER and BER were also important in protecting cells from platinum-induced cytotoxicity16. It was suggested that maintaining
transcriptional integrity is more crucial for cell survival upon oxaliplatin exposure than GG-NER16. This hypothesis was confirmed by conducting a cell viability assays, which showed that cells deficient in TC-NER were more sensitive to oxaliplatin than GG-NER deficient cells. Lans also found that oxaliplatin and cisplatin treatment caused an increase in cellular ROS levels, and BER is initiated by the oxidative damage from platinum drug exposure, suggesting that BER mainly protects platinum drug-induced cytotoxicity by removing oxidative DNA damage rather than acting on oxaliplatin DNA crosslink16. The developed assay can be used to elucidate the involvement of various DNA damage pathways in the recognition and repair of oxaliplatin DNA crosslinks, for example, by knocking out a protein of interest, disrupting the repair pathways where this protein is involved and then looking at the distribution of oxaliplatin-DNA adducts.
TLS polymerases can affect cancer therapy by synthesizing DNA opposite to lesions and inducing mutations in important genomic regions. It is known that multiple TLS polymerases are involved in bypassing lesions formed by platinum drugs. The efficiency and fidelity of bypass by these TLS polymerases can affect the mutagenicity of the adducts, and it is unknown how different TLS
polymerases mediate resistance to platinum drugs. Therefore, TLS polymerases can be manipulated in a way to influence tumorigenesis and enhance cancer treatment outcomes31. For cisplatin-induced crosslinks, it was found that Pol η and Pol ζ are cooperating when synthesizing DNA opposite the adduct. This depends on the activity of TLS polymerase REV1, which is important in polymerase switching during TLS32. Canman suggests that Pol η is not crucial in the bypass of oxaliplatin, satraplatin, and picoplatin when compared with REV1 and Pol in the context of two different cancer cell lines33. Canman also highlights that since Pol κ can perform error-free bypass of bulkier adducts such as benzo[α]pyrene, and that Pol κ can bypass adducts formed by oxaliplatin during DNA replication. Regardless of lesion type, Pol η and Pol κ still require Pol ζ to extend beyond nucleotides inserted opposite DNA adducts during TLS with the help of REV133. In addition, recent data suggest that cisplatin and cyclophosphamide resistance are associated with REV1 and REV3 activities in B- cell lymphoma and lung adenocarcinoma, and depletion of REV3 and REV1 caused an increase in sensitivity of those tumors to drugs and reduced mutagenesis rate34, 35. These results suggest that targeting REV1 and Pol ζ can have a synergistic anti-cancer effect, and a similar approach might be applied to other platinum-based agents such as oxaliplatin, carboplatin, satraplatin, and picoplatin.
![Figure 6. Representative fragmentation pattern of OXP-d(GG) analyte. For OXP-d(GG), the major fragments after PRM experiments were [Gua-Pt(NH 3 )] + and [Gua-Pt(NH 3 )-Gua] +](https://thumb-ap.123doks.com/thumbv2/123dokinfo/10494294.0/24.893.117.779.107.413/figure-representative-fragmentation-pattern-analyte-major-fragments-experiments.webp)
