A phase I trial of ABT-888 (veliparib), a poly(ADP-ribose) polymerase (PARP inhibitor), in combination with topotecan, a topoisomerase I–targeted agent, was performed to determine maximum tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of the combination in patients with refractory solid tumors and lymphomas. Varying schedules and doses of intravenous topotecan in combination with ABT-888 (10 mg) administered orally twice a day (BID) were evaluated. Plasma and urine pharmacokinetics were assessed, and levels of poly(ADP-ribose) (PAR) and the DNA-damage marker, γH2AX, were measured in tumor and peripheral blood mononuclear cells (PBMCs). Twenty-four patients were enrolled. Significant myelosuppression limited the ability to co-administer ABT-888 with standard doses of topotecan, necessitating dose reductions. Preclinical studies using athymic mice carrying human tumor xenografts also informed schedule changes. The MTD was established as topotecan 0.6 mg/m2/day and ABT-888 10 mg BID on days 1–5 of 21-day cycles. Topotecan did not alter the pharmacokinetics of ABT-888. A more than 75% reduction in PAR levels was observed in 3 paired tumor biopsy samples; a greater than 50% reduction was observed in PBMCs from 19 of 23 patients with measurable levels. Increases in γH2AX response in circulating tumor cells (CTC) and PBMCs were observed in patients receiving ABT-888 with topotecan. We demonstrate a mechanistic interaction of a PARP inhibitor, ABT-888, with a topoisomerase I inhibitor, topotecan, in PBMCs, tumor, and CTCs. Results of this trial reveal that PARP inhibition can modulate the capacity to repair topoisomerase I–mediated DNA damage in the clinic.

Purpose: The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties. Experimental Design: Tumor fragments from tumor-bearing MMTV-PyMTor cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53 +/À , BRCA1/p53, and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naI« ve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors. Results: PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/ p53 +/À and wnt1/p53 +/À , and 15% with Her2/neu tumors. Expansion of PyMTand wnt1tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel. Conclusions: Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

BackgroundDevelopment of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial.ResultsForty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment.ConclusionsThis study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.Electronic supplementary materialThe online version of this article (doi: 10.1186/1471-2164-15-393) contains supplementary material, which is available to authorized users.

BackgroundThe nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.MethodsStructure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays.ResultsResults demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance.ConclusionOverall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

BackgroundTopoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors.Methodology/Principal FindingsWe have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998.Conclusions/SignificanceOur analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.

The anticancer activity of 5’-sulfamoyl-purines in cell culture has been previously reported, but there are no published investigations of activity in vivo. One of the earliest members of this class, 5’-O-aminosulfonyl-adenosine, NSC133114, was reported to have 200nM IC50 in L1210 cells (A.Bloch, Biochemistry 10:4394, 1971). We found the average GI50 for NSC133114 was 10 nM across the full NCI-60 cell line panel. When tested in vivo, NSC 133114 was active in the NCI hollow fiber assay, with an overall score of 28 (maximum possible score is 96). NSC750854, the 6-desamino derivative of NSC133114, also had an average GI50 of 10 nM in the NCI-60 panel, but it was nearly twice as active in vivo as NSC133114, with a hollow fiber score of 52. Thus, NSC750854 was chosen for more extensive testing in a set of 8 distinct xenografts. The A498 human renal tumor was the most sensitive xenograft, responding to NSC 750854 via the PO, IP and IV routes. Complete regressions of tumors and tumor-free animals at the end of the study were produced by NSC750854 when administered IP once daily at 5 mg/kg for 2 cycles of 5 days or 3.75 mg/kg twice daily (BID) for 2 cycles of 5 days. At an oral dose of 5 mg/kg given BID for 2 cycles of 6-10 doses each, NSC 750854 produced A498 tumor stasis in the absence of body weight loss. Administered IV at 6.4 mg/kg every other day for 2 cycles of 5 doses or 3.2 mg/kg BID every other day for 2 cycles of 10 doses, NSC 750854 also produced tumor regressions, but no tumor free animals at the end of the study. RPMI-8226 myeloma was the xenograft with the next most sensitive response to NSC750854, with durable tumor stasis following administration by all 3 routes. NSC750854 produced tumor stasis and some regressions in the U251 CNS tumor xenograft, but they were more transient. The PC-3 prostate and LOX melanoma xenografts were responsive to NSC750854 with tumor growth stasis of varying durations. NSC 750854 was effective by all 3 routes of administration but the most dramatic activity was noted with the IP dose route. Among the 8 models tested, the least responsive tumors under the conditions evaluated were Colo 205 colon, HCT-15 colon and UACC-62 melanoma. NSC750854 produced tumor growth suppression but no durable tumor stasis in these 3 models. These patterns of activity in vitro and in vivo for NSC750854 are substantially different than the patterns observed for the anticancer purines that are approved for clinical use. Ongoing studies with NSC 750854 include determinations of activity in advanced tumors, pediatric human tumor models, pharmacologic behavior, toxicologic profile in additional species, and mechanisms of action. This work was conducted under an approved IACUC protocol in AAALACi accredited facilities and supported by federal funds from the National Cancer Institute, NIH, under Contract No. HHSN261200800001E. Citation Format: Melinda G. Hollingshead, Michelle Gottholm-Ahalt, Howard Stotler, John Carter, Cheryl Domitrovich, Adrienne Horner, Jerry M. Collins. 5’-Sulfamoyl purines are highly active in many xenografts of human solid tumors via oral, intravenous, and intraperitoneal routes of delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2013-4489

In this article, 5-aza-4 0 -thio-2 0 -b-fluoro-2 0 -deoxycytidine (F-aza-T-dCyd, NSC801845), a novel cytidine analog, is first disclosed and compared with T-dCyd, F-T-dCyd, and aza-T-dCyd in cell culture and mouse xenograft studies in HCT-116 human colon carcinoma, OVCAR3 human ovarian carcinoma, NCI-H23 human NSCLC carcinoma, HL-60 human leukemia, and the PDX BL0382 bladder carcinoma. In three of five xenograft lines (HCT-116, HL-60, and BL-0382), F-aza-T-dCyd was more efficacious than aza-T-dCyd. Comparable activity was observed for these two agents against the NCI-H23 and OVCAR3 xenografts.In the HCT-116 study, F-aza-T-dCyd [10 mg/kg intraperitoneal (i.p.), QDx5 for four cycles], produced complete regression of the tumors in all mice with a response that proved durable beyond postimplant day 150 (129 days after the last dose). Similarly, complete tumor regression was observed in the HL-60 leukemia xenograft when mice were dosed with F-aza-T-dCyd (10 mg/kg i.p., QDx5 for three cycles). In the PDX BL-0382 bladder study, both oral and i.p. dosing of F-aza-T-dCyd (8 mg/kg

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