John A. Hartley

EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment.

Epidermal growth factor receptor (EGFR) overexpression is associated with resistance to chemotherapy and radiotherapy. It modulates DNA repair after radiation-induced damage through association with the catalytic subunit of DNA protein kinase (DNA-PKcs). We investigated the role of EGFR nuclear import and its association with DNA-PKcs on DNA repair after exposure to cisplatin or ionizing radiation (IR). The model system was based on EGFR-null murine NIH3T3 fibroblasts in which EGFR expression was restored with isoforms that were wild-type (wt), derived from human cancers (L858R, EGFRvIII), or mutated in the nuclear localization signal (NLS) sequence. In cells expressing wtEGFR or EGFRvIII, there was complete unhooking of cisplatin-induced interstrand cross-links and repair of IR-induced strand breaks. In contrast, cells expressing L858R or NLS mutations showed reduced unhooking of interstrand cross-links and repair of strand breaks. Immunoprecipitation showed wtEGFR and EGFRvIII binding to DNA-PKcs, increasing 2-fold 18 hours after cisplatin therapy. Confocal microscopy and proximity ligation assay showed that this interaction in the cytoplasm and nucleus was associated with increased DNA protein kinase complex (DNA-PK) activity. Cells expressing the EGFR L858R mutation, which has constitutive kinase activity, exhibited reduced DNA repair without nuclear localization. EGFR-NLS mutants showed impaired nuclear localization and DNA-PKcs association with reduced DNA repair and DNA-PK kinase activity. In summary, EGFR nuclear localization was required for modulation of cisplatin and IR-induced repair of DNA damage. EGFR-DNA-PKcs binding was induced by cisplatin or IR but not by EGFR nuclear translocation per se. Our findings show that EGFR subcellular distribution can modulate DNA repair kinetics, with implications for design of EGFR-targeted combinational therapies.

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.

SJG-136 (NSC 694501), A Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 2: Efficacy Evaluations

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.

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.

SG2285, a Novel C2-Aryl-Substituted Pyrrolobenzodiazepine Dimer Prodrug That Cross-links DNA and Exerts Highly Potent Antitumor Activity

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents

HER2 (ErbB2) is overexpressed in up to 30% of human breast cancers. Preclinical and clinical studies suggest synergy between some chemotherapeutic agents and the humanized anti-HER2 antibody trastuzumab (Herceptin). This study investigated the effects of etoposide and cisplatin on the repair of DNA damage in breast cancer cell lines. We examined the potential significance of HER2 nuclear expression in DNA repair. MCF-7, SK-BR-3, and MDA-MB-453 cells were treated with cisplatin and etoposide. Repair of DNA interstrand crosslinks (ICL) and strand breaks, following incubation with cisplatin and etoposide, respectively, were quantitated by the single-cell gel electrophoresis (comet) assay. Intrastrand crosslinks produced by cisplatin were assessed by ELISA. The effects of trastuzumab were measured in combination with these drugs. Similar experiments were done using HER2-negative MDA-MB-468 cells transfected with HER2 and a construct lacking the nuclear localization sequence. Incubation of breast cancer cell lines with trastuzumab delayed the repair of ICL produced by cisplatin. There were no effects on the repair of intrastrand crosslinks produced by cisplatin, or repair of DNA strand breaks following etoposide treatment. Transfection of HER2 into MDA-MB-468 cells inhibited the repair of cisplatin-induced ICL, whereas transfection of a HER2 construct lacking the nuclear localization sequence did not affect DNA repair. These results indicate that HER2 expression modulates the repair of specific DNA lesions produced by chemotherapy. The effect on ICL repair requires nuclear expression of HER2. Understanding the mechanisms of interaction between DNA-interacting agents and HER2 inhibitors will inform the design of clinical trials and optimize the therapeutic effects of these combinations. [Mol Cancer Ther 2009;8(11):3015–23]

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.

DNA interstrand cross-linking and in vivo antitumor activity of the extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057

SummaryThe pyrrolobenzodiazepines (PBDs) are naturally occurring antitumor antibiotics and a PBD dimer (SJG-136, SG2000) is in Phase II trials. SG2000 is a propyldioxy linked PBD dimer which binds sequence selectively in the minor groove of DNA forming DNA interstrand and intrastrand cross-linked adducts, and also mono-adducts depending on sequence. SG2057 is the corresponding dimer containing a pentyldioxy linkage. SG2057 has multilog differential in vitro cytotoxicity against a panel of human tumour cell lines with a mean GI50 of 212 pM. The agent is highly efficient at producing DNA interstrand cross-links in cells which form rapidly and persist over a 48xa0h period. Significant antitumor activity was demonstrated in several human tumor xenograft models. Cures were obtained in a LOX-IMVI melanoma model following a single administration and dose-dependent activity, including regression responses, observed in SKOV-3 ovarian and HL-60 promyelocytic leukemia models following repeat dose schedules. In the advanced stage LS174T model, SG2057 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. SG2057 is therefore a highly active antitumor agent, with more potent in vitro activity and superior in vivo activity to SG2000, warranting further development.

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant’ cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death.

Quinone Oxidoreductase-2-Mediated Prodrug Cancer Therapy

An oxidoreductase enzyme, abundant in tumors, can be catalytically activated by a synthetic cofactor to convert a prodrug to a cytotoxic DNA binding species in human subjects. A Lethal Combination for Liver Cancer? Undergoing chemotherapy to treat cancer is a dreaded—and often grueling—experience. Many chemotherapeutic drugs are double-edged swords: Used with the aim of destroying rapidly dividing cancer cells, they also kill normal cells that exhibit this behavior, sometimes resulting in debilitating side effects. For example, destruction of the quickly multiplying cells that line the gastrointestinal tract can lead to painful ulcerations; loss of proliferating bone marrow cells reduces immune system function and can cause anemia. Ideally, chemotherapeutic drugs display greater specificity toward cancer cells, thus reducing toxicity to healthy tissue. Now, Middleton and colleagues describe their work on a chemotherapy regime that shows promise for specifically destroying liver cancer. The system in question is based on the weak alkylating agent CB1954. Monofunctional alkylating agents such as CB1954 react with a single DNA residue, whereas the more potent bifunctional alkylating agents can react with two residues on different DNA strands, cross-linking them and preventing the strands from uncoiling during replication. Rapidly proliferating cells that duplicate their DNA frequently are more sensitive than others to such damage. CB1954 can be converted to a far more cytotoxic bifunctional agent by the oxidoreductase enzyme NQO2—which is ubiquitously expressed in human tissues—but only in the presence of a cofactor. Middleton and co-workers investigated how this chemotherapy system might be used most effectively. Using structural data, they modeled how CB1954 and a synthetic cofactor bind to NQO2, which indicated that the cofactor should saturate NQO2 before CB1954 is added to maximize conversion of CB1954 to its cytotoxic form. On the basis of these data, a phase I clinical study was performed, during which first the cofactor and then CB1954 were administered to patients with several types of cancer, and a maximum tolerated dose for the drug combination was determined. As predicted, DNA cross-links were observed in tumors after treatment. The researchers also compared the fates of the cofactor and CB1954 when they were administered separately or in combination, and saw a striking interaction: The plasma concentration of CB1954 dropped much more sharply in the presence than in the absence of the cofactor, indirectly indicating activation of NQO2 by the cofactor. However, side effects from the drug combination were similar to those caused by CB1954 alone. NQO2 activity was generally elevated in tumors and was low in bone marrow, blood cells, and epithelial cells from the colon, providing an explanation for the lack of additional side effects caused by use of the cofactor. NQO2 activity in liver cancer was higher than in other cancers by a factor of 6, suggesting that the CB1954-cofactor drug combination might be especially effective for treating liver cancer. Moreover, normal liver cells are relatively resistant to the effects of DNA cross-links, probably because of their low rates of cell division. Thus, the authors propose that a phase II efficacy trial for liver cancer patients is in order for this chemotherapeutic agent. DNA-damaging agents are widely used in cancer treatment despite their lack of tumor specificity. Human NQO2 (quinone oxidoreductase-2) is an atypical oxidoreductase because no endogenous electron donor has been identified to date. The enzyme converts CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], in the presence of the synthetic nicotinamide cofactor analog EP0152R, to a cytotoxic bifunctional alkylating agent. NQO2 activity in hepatocellular tumor tissue is higher than that in other cancer types by a factor of 6 and higher than that in bone marrow by a factor of 20. Structural data from x-ray crystallography and nuclear magnetic resonance spectroscopy allowed us to construct a model of CB1954 and EP0152R binding to NQO2, which suggested an optimal infusion schedule for a phase I trial combining the two agents. Thirty-two patients were treated, and diarrhea and serum transaminase concentrations defined a maximum tolerated dose for the drug combination. There was a clear pharmacokinetic interaction, with EP0152R inducing a marked increase in clearance of CB1954, in keeping with model predictions. We detected DNA interstrand cross-links caused by nitroreduced CB1954 in tumor biopsies from treated patients, demonstrating that the activated prodrug exerts its cytotoxic properties through DNA base alkylation.