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Summary Background: The DNA repair protein O 6 -alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitrosoureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloroethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine. Patients and methods: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m 2 , divided over two days. Fotemustine 100 mg/m 2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics. Results: The maximum tolerated dose (MTD) of TMZ was 400 mg/m 2 (200 mg/m 2 /d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs. Conclusions: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF–induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation. Cancer Res; 71(4); 1396–405. ©2011 AACR.

We evaluated the effects of the trinuclear platinum complex, BBR 3464, in a human ovarian carcinoma cell line (OAW42) and in its cisplatin (CDDP)-resistant counterpart (OAW42MER). A 14-fold increased sensitivity to a 1-h BBR 3464 exposure was found in OAW42MER cells compared with their parental cell line. Flow cytometric experiments showed that BBR 3464 was able to induce a persistent block of OAW42 and OAW42MER cells in the G 2 M phase, whereas CDDP caused an initial accumulation of cells in the S phase followed by an increase in the G 2 M cell fraction in both cell lines. Exposure to equitoxic (IC 50 ) drug concentrations induced programmed cell death in both cell lines. However, the percentage of cells with an apoptotic nuclear morphology was slightly higher after CDDP than BBR 3464 treatment in OAW42 cells, whereas the opposite pattern was observed in OAW42MER cells. Degradation of the nuclear lamin B was detected in OAW42 cells after exposure to each drug. Conversely, in OAW42MER cells lamin B cleavage was only appreciable after BBR 3464 exposure. In OAW42 cells, CDDP and BBR 3464 did not appreciably affect the mitochondrial membrane potential (Δψ mt ), whereas in the OAW42MER cell line a marked Δψ mt reduction was observed after exposure to BBR 3464, but not to CDDP. The results of the study would suggest that the sensitivity to BBR 3464 observed in the CDDP-resistant OAW42MER cell line might be attributable to the ability of the trinuclear platinum complex to modify DNA in a way which is different from that of CDDP and, as a consequence, to induce different cellular responses to DNA damage such as the triggering of specific apoptotic pathways.

Mismatch DNA repair deficiency is associated with resistance to certain major groove alkylating agents including methylating agents and cisplatin. We have now studied the relevance of mismatch repair alterations to the cytotoxicity induced by drugs which alkylate N3 adenines in the minor groove of DNA. We have used the mismatch repair defective human colocarcinoma cell line HCT-116 which has a mutation in the hMLH1 gene, and a subline where hMLH1 expression is restored by chromosome 3 transfer (HCT-116+ch3). We have tested three alkylating minor groove binders (tallimustine, carzelesin and CC1065) and one non-covalent minor groove binder (PNU 151807). The HCT-116+ch3 subline was more sensitive than the parental line to the treatment with the three alkylating minor groove binders, while the non-alkylating compound had a similar activity in both cell lines. Further support for mismatch repair being involved in sensitivity of the minor groove alkylators is that two cisplatin-resistant sublines of the human ovarian adenocarcinoma cell line A2780 (A2780/CP70 and A2780/MCP-1) are defective in hMLH1 expression and are more resistant to these agents than the parental mismatch repair proficient cells. Furthermore, the restoration of hMLH1 activity in the A2780/CP70 cell line, by introduction of chromosome 3, was associated with an increased sensitivity to the three alkylating minor groove binders. Again, the non-covalent minor groove binder was equally effective in mismatch repair deficient and proficient clones. The data indicate that mismatch repair deficiency mediated by loss of hMLH1 expression is associated not only with drug-resistance to major groove binders, but also to minor groove binders. However, loss of mismatch repair does not mediate resistance to the non-covalent minor groove binder PNU 151807. © 1999 Cancer Research Campaign

E-3810 is a novel small molecule that inhibits VEGF receptor-1, -2, and -3 and fibroblast growth factor receptor-1 tyrosine kinases at nmol/L concentrations currently in phase clinical II. In preclinical studies, it had a broad spectrum of antitumor activity when used as monotherapy in a variety of human xenografts. We here investigated the activity of E-3810 combined with different cytotoxic agents in a MDA-MB-231 triple-negative breast cancer xenograft model. The molecule could be safely administered with 5-fluorouracil, cisplatin, and paclitaxel. The E-3810–paclitaxel combination showed a striking activity with complete, lasting tumor regressions; the antitumor activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel+brivanib and paclitaxel+sunitinib. Pharmacokinetics studies suggest that the extra antitumor activity of the combination is not due to higher paclitaxel tumor levels, which in fact were lower in mice pretreated with all three kinase inhibitors, and the paclitaxel plasma levels excluded reduced drug availability. Pharmacodynamic studies showed that E-3810, brivanib, and sunitinib given as single agents or in combination with paclitaxel reduced the number of vessels, but did not modify vessel maturation. Reduced tumor collagen IV and increased plasma collagen IV, associated with increased matrix metalloproteinases (MMP), particularly host MMP-9, indicate a proteolytic remodeling of the extracellular matrix caused by E-3810 that in conjunction with the cytotoxic effect of paclitaxel on the tumor cells (caspase-3/7 activity) may contribute to the striking activity of their combination. These data support the therapeutic potential of combining E-3810 with conventional chemotherapy. Mol Cancer Ther; 12(2); 131–40. ©2012 AACR.

A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N- ras) level were observed between BBR 3464 and CDDP. © 2001 Cancer Research Campaign www.bjcancer.com

E-3810 is a small molecule inhibitor of VEGFR-1, -2 and -3 and FGFR-1 tyrosine kinases with IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C192.

E-3810 is a novel small molecule that selectively inhibits VEGF receptor-1, -2 and -3 and FGF receptor-1 (FGFR-1) tyrosine kinases currently undergoing a Phase 1 clinical trial in Europe. We have previously shown that the compound has strong anti-angiogenic effects in vivo and displays potent activity in all the human xenograft models in which it has been tested. We have also recently demonstrated that interference with FGF/FGFR pathway could result in a higher drug cytotoxic activity in those cell lines whose growth in vitro was dependent on the pathway. In order to corroborate these findings in an in vivo setting, a panel of human xenografts characterized by different expression of FGFR-1and FGF2 ligand is being screened for the response to E-3810 given at its optimal dose (20 mg/kg per os daily). Athymic female mice were transplanted s.c. with the different human xenografts and, when tumor masses reached the size of 80-120 mg, were randomized to receive vehicle or E-3810 for 14 days. Results are available for 7 models (see table reported below). All the selected tumors displayed similar growth rates, except for RXF_486 that grew more slowly. Tumor growth resumed upon drug withdrawal, at a rate seemingly higher where FGFR-1 was highly expressed. These preliminary results confirm the strong activity of E-3810 in a large panel of tumours, with growth control and, sometimes, regressions. Studies are ongoing to enlarge the panel of models and to confirm the observed trend suggesting a somewhat higher sensitivity to E-3810 in tumors expressing higher FGFR-1 and\or FGF ligand. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 595. doi:10.1158/1538-7445.AM2011-595

E-3810 is a novel small molecule that selectively inhibits VEGFR-1, -2 and -3 and FGFR-1 tyrosine kinases with IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2574. doi:10.1158/1538-7445.AM2011-2574

E-3810, 6-[[7-[(1-aminocyclopropyl)methoxy]-6-methoxy-4-quinolyl]oxy]-N-methyl-naphthalene-1-carboxamide, is a novel small molecule that potently and selectively inhibits VEGFR1-3 and FGFR-1 tyrosine kinases (TKs) with IC50 in the 10-20 nM range. Concentrations at least 1-order higher were required to inhibit FGFR-2 and 3, PDGFRα and β and c-Kit receptors and no relevant activity was seen against different cancer-related TKs. E-3810 1-10 nM inhibited the ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 and the downstream phosphorylation of Erk in HUVEC cells; inhibition of PDGFR- auto-phosphorylation in NIH-3T3 cells was detected at concentrations higher than 100 nM. E-3810 inhibited the VEGF- and FGF-driven growth of HUVEC cells (IC50 40-50 nM), while anti-proliferative activity on different human cancer cell lines was shown at 10-30 M concentrations. E-3810 was tested in different human tumor xenografts in nude mice, including colon HT-29, ovarian A-2780, renal A498, RXF393 and SKN12I carcinomas. A dose dependent antitumor effect was observed in a range from 10 to 40 mg/kg given orally for 30 consecutive days. The optimal dose schedule, 20 mg/kg for 30 days, was well tolerated and caused up to 90% suppression of tumor growth. Sustained growth inhibition was also seen when treatment was repeated after a 2-week dosing interval or when dosing was initiated at a larger tumor volume (average 400 mm3). Tumor regression was observed in the RXF393 xenograft. E-3810 showed antitumor activity equal or better than Sunitinib and Brivanib at their respective optimal doses. Pharmacokinetic studies in mice demonstrated high oral bioavailability, high volume of distribution and terminal half life of 4 hrs. After single and repeated administration E-3810 systemic exposure was proportional to the dose, without any accumulation after repeated doses. The good bioavailability and the remarkable antitumor activity of E-3810 in several tumor xenografts make this compound an interesting candidate for clinical development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A257.

Some DNA minor groove binders have shown antiviral and antitumor activity. The distamycin derivative Tallimustine and the CC-1065 derivative Adozelesin and Carzelesin are under clinical investigationin phase II clinical trials. The mechanisms of repair of the DNA lesions caused by these drugs are unknown. By using the gel retardation assay, we have identified a protein able to recognize a fragment of DNA only if it was previously reacted with minor groove binders (MGB). This protein binds with very high affinity AT containing DNA treated with MGB such as Distamycin A or Hoechst 33258 but not with a major groove binders (such as e.g. quinacrine mustard). This protein was found to be present in extracts of human, murine or hamster cells, although the human protein appears to have a molecular weight slightly lower than that of the other species. This protein is expressed both in cancer and normal tissues. By using ultrafiltration techniques as well as Southwestern analysis it was estimated that the apparent molecular weight is close to 100 Kda. Work is in progress to purify this protein which might be involved in the recognition and repair of DNA damage caused by minor groove binders.

Background Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera. Methods We characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP). Results JN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis. Conclusions The JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3091-1) contains supplementary material, which is available to authorized users.