Victoria J. Spanswick

Repair of DNA interstrand crosslinks: molecular mechanisms and clinical relevance

Drugs that produce DNA interstrand crosslinks (ICLs), between the two complementary strands of the double helix, have an important role in chemotherapy regimens for cancer. Novel crosslinking agents, and targeting strategies involving DNA crosslinking agents, continue to be developed. The ability of cells to repair DNA ICLs is a critical determinant of sensitivity, and recent dinical studies indicate that DNA repair capacity is strongly implicated in both inherent tumour sensitivity and acquired drug resistance. A detailed understanding of the cellular mechanisms that act to eliminate these critical DNA lesions is clearly important. DNA ICLs present a complex challenge to DNA repair mechanisms because of the involvement of both DNA strands. It is now clear that cells from bacteria and yeast to mammals eliminate interstrand ICLs through the coordinated action of several DNA repair pathways. Recently, a model of ICL repair has been proposed, in which mammalian cells use novel excision repair reactions (requiring the XPF and ERCC1 proteins) to uncouple the crosslink. This is followed by a homologous recombination step to provide the genetic information needed to complete repair. This new knowledge may permit the development of screens for tumour response to crosslinking agents, and should also aid the design of more effective crosslinking agents that evade DNA repair. In addition, the proteins mediating the repair reactions represent potential targets for therapeutic intervention.

SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 1: Cellular Pharmacology, In vitro and Initial In vivo Antitumor Activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.

Phase I study of sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors.

Purpose: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. Experimental Design: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 μg/m2) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 μg/m2 due to unexpected toxicity. Results: The maximum tolerated dose of SJG-136 was 45 μg/m2. The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive γ-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. Conclusions: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

γ-H2AX Foci Formation as a Pharmacodynamic Marker of DNA Damage Produced by DNA Cross-Linking Agents: Results from 2 Phase I Clinical Trials of SJG-136 (SG2000)

Purpose: To evaluate γ-H2AX foci as a pharmacodynamic marker for DNA damage induced by DNA interstrand cross-linking drugs. Experimental Design: γ-H2AX foci formation was validated preclinically in comparison with the Comet assay, and evaluated pharmacodynamically in two phase I studies of different dosing schedules of the novel cross-linking agent SJG-136 (SG2000). Results: The measurement of γ-H2AX foci in human fibroblasts and lymphocytes in vitro was more than 10-fold more sensitive than Comet assay measurement of cross-linking, with peak γ-H2AX response 24 hours after the peak of cross-linking. In lymphocytes from a phase I study (every three week schedule), γ-H2AX foci were detectable 1 hour following the end of administration, and in all patients, maximum response was observed at 24 hours. Significant levels of foci were still evident at days 8 and 15 consistent with the known persistence of the DNA damage produced by this agent. In two tumor biopsy samples, foci were detected 4 hours postinfusion with levels higher than in lymphocytes. Extensive foci formation was also observed before the third dose in cycle 1 in lymphocytes from a second phase I study (daily × 3 schedule). These foci also persisted with a significant level evident before the second cycle (day 21). An increased γ-H2AX response was observed during the second cycle consistent with a cumulative pharmacodynamic effect. No clear relationship between foci formation and administered drug dose was observed. Conclusion: This is the first use of γ-H2AX as a pharmacodynamic response to a DNA cross-linking agent in a clinical trial setting. Clin Cancer Res; 19(3); 721–30. ©2012 AACR.

Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells

We have studied the actions of autologous plasma on both basal and DNA damage‐induced apoptosis in B‐chronic lymphocytic leukaemia (B‐CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis‐specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and γ‐radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons α or γ or stromal cell‐derived factor 1, each of which have been shown to protect B‐CLL cells from apoptosis in vitro. Plasma addition to B‐CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti‐apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3′‐kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B‐CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective‐signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B‐CLL treatment.

Measurement of DNA Interstrand Crosslinking in Individual Cells Using the Single Cell Gel Electrophoresis (Comet) Assay

The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.

Inhibition of Carboplatin-Induced DNA Interstrand Cross-link Repair by Gemcitabine in Patients Receiving these Drugs for Platinum-Resistant Ovarian Cancer

Background: The potential of gemcitabine to interact with carboplatin was explored in a phase II trial in platinum-resistant ovarian cancer. Peripheral blood lymphocytes were sampled after drug administration to measure DNA interstrand cross-link formation and repair. Patients and Methods: Forty patients received carboplatin target area under concentration-time curve (AUC 4) followed by gemcitabine 1,000 mg/m2 with a second dose of gemcitabine on day 8. Peripheral blood lymphocytes were obtained in 12 patients before and at intervals during the first cycle of chemotherapy. DNA cross-link formation and repair (unhooking) were measured by the single-cell gel electrophoresis (comet) assay following ex vivo incubation. Results: The global response rate was 47% (Response Evaluation Criteria in Solid Tumors rate, 29%; CA125 rate, 63%). Delays in treatment were seen in 24% of cycles largely due to myelosuppression; 15% of day 8 administration was omitted. Peak carboplatin-induced DNA cross-linking was seen by 24 hours. Significant reduction was seen in the repair of in vivo carboplatin-induced DNA cross-links following administration of gemcitabine. Conclusion: An enhanced activity of carboplatin in platinum-resistant ovarian cancer may be due to synergy with gemcitabine through inhibition of repair of DNA cross-links. Future studies should explore coadministration of these drugs, as this may be a more effective schedule. Clin Cancer Res; 16(19); 4899–905. ©2010 AACR.

Measurement of drug-induced DNA interstrand crosslinking using the single-cell gel electrophoresis (comet) assay.

DNA damaging agents have been widely used in cancer chemotherapy for many years and have proved successful in the treatment of both solid tissue and haematological malignancies. Many commonly used clinical agents, such as members of the nitrogen mustard, chloroethylnitrosourea, dimethane-sulphonate and platinum classes, are bifunctional. DNA interstrand crosslinks (ISC) formed in cells are clearly critical cytotoxic lesions and the formation of DNA ISC has been shown to correlate with cytotoxicity in vitro (1-5). Acquired resistance in vitro to such agents can occur by a number of mechanisms, for example altered drug transport (6), intracellular detoxification via enhanced glutathione and glutathione-S-transferase activity (7), but enhanced DNA repair capacity can also play an important role (3). Clinically the mechanisms of acquired resistance to DNA damaging agents are less clear but enhanced repair of ISC has been suggested to play a role in the acquired resistance of some cancers, e.g., chronic lymphocytic leukaemia to nitrogen mustards (8). In addition, the inherent sensitivity (and curability) of some tumors, e.g., testicular cancer, to DNA damaging agents may result in part from their inability to repair critical DNA lesions (9).

Phase I Pharmacokinetic and Pharmacodynamic Study of SJG-136, a Novel DNA Sequence Selective Minor Groove Cross-linking Agent, in Advanced Solid Tumors

Purpose: This phase I study assessed the maximum tolerated dose (MTD), safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of SJG-136, a sequence-specific DNA cross-linking agent, in patients with advanced cancer. Experimental Design: In schedule A, seven patients received escalating doses of SJG-136 (6, 12, 24, and 48 μg/m2) daily for 5 of 21 days. Blood samples were collected for PK analysis on days 1 and 5 of cycle 1. In schedule B, SJG-136 was given daily for 3 of 21 days (N = 17; doses 20, 25, 30, and 35 μg/m2). Blood samples were collected on days 1 and 3 of cycles 1 and 2 for PK and PD analysis. Patients in schedule B received dexamethasone and early diuretic care. Results: Schedule A—dose-limiting toxicities included grade 3 edema, dyspnea, fatigue, and delayed liver toxicity (grade 3–4). PK analysis revealed dose-dependent increases in AUC and Cmax. Substantial changes in volume of distribution at steady-state occurred after repeated dosing in some patients prior to the onset of edema. Schedule B—the same toxicities were manageable with steroid premedication and diuretic support. No significant myelosuppression occurred on either schedule. DNA interstrand cross-links correlated with systemic exposure of SJG-136 following the second dose in cycle 1 and were still detectable immediately before cycle 2. Conclusions: The MTD of SJG-136 in this study was 30 μg/m2 administered on a daily 3× basis with no myelosuppression effects. Coupled with supportive management, SJG-136 is now advancing to a phase II trial in ovarian cancer. Clin Cancer Res; 17(11); 3794–802. ©2011 AACR.

Time-dependent cytotoxicity induced by SJG-136 (NSC 694501): influence of the rate of interstrand cross-link formation on DNA damage signaling

SJG-136 is a new pyrrolobenzodiazepine dimer inducing time-dependent cytotoxicity. HCT 116 cells were exposed to 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours to achieve similar levels of interstrand cross-links (ICL). The short exposure led to a rapid formation of ICLs (1 hour), early H2AX foci formation (4 hours), prominent S phase arrest, and greater phosphorylation of Nbs1 (on serine 343) and Chk1 (on serine 317) than a 24-hour exposure. The prolonged exposure at low concentrations of SJG-136 induced a gradual formation of ICLs (up to 24 hours) which was associated with a limited S phase arrest and delayed Nbs1 phosphorylation. Prolonged exposure was also associated with a reduced phosphorylation of p53 on serines 15 and 20, a limited and delayed phosphorylation on serine 392, and a less prominent increase in p21 levels. These data suggest that the 24-hour exposure to a low concentration of SJG-136 led to delayed and reduced DNA damage signaling compared with a higher concentration of SJG-136 for 1 hour, resulting in greater cytotoxicity and contributing to the time-dependent cytotoxic effect of SJG-136. [Mol Cancer Ther 2006;5(6):1602–9]