BackgroundPatients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered.Methods and FindingsWe postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups).ConclusionsThe novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.

Purpose Identifying novel therapeutic agents for the treatment of childhood cancers requires preclinical models that recapitulate the molecular characteristics of their respective clinical histotypes. Experimental Design and Results Here, we have applied Affymetrix HG-U133Plus2 profiling to an expanded panel of models in the Pediatric Preclinical Testing Program. Profiling led to exclusion of two tumor lines that were of mouse origin and five osteosarcoma lines that did not cluster with human or xenograft osteosarcoma samples. We compared expression profiles of the remaining 87 models with profiles from 112 clinical samples representing the same histologies and show that model tumors cluster with the appropriate clinical histotype, once “immunosurveillance” genes (contributed by infiltrating immune cells in clinical samples) are eliminated from the analysis. Analysis of copy number alterations using the Affymetrix 100K single nucleotide polymorphism GeneChip showed that the models have similar copy number alterations to their clinical counterparts. Several consistent copy number changes not reported previously were found (e.g., gain at 22q11.21 that was observed in 5 of 7 glioblastoma samples, loss at 16q22.3 that was observed in 5 of 9 Ewing’s sarcoma and 4 of 12 rhabdomyosarcoma models, and amplification of 21q22.3 that was observed in 5 of 7 osteosarcoma models). We then asked whether changes in copy number were reflected by coordinate changes in gene expression. We identified 493 copy number – altered genes that are nonrandom and appear to identify histotype-specific programs of genetic alterations. Conclusions These data indicate that the preclinical models accurately recapitulate expression profiles and genetic alterations common to childhood cancer, supporting their value in drug development.

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Deregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly mTOR inhibitors such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic agents. Inhibition of mTOR results in sensitization to DNA damaging agents, however the molecular mechanism is not well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhanced sensitivity of a pediatric rhabdomyosarcoma xenograft toradiotherapy and sensitized rhabdomyosarcoma cells to interstrand crosslinker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia (FA) pathway, FANCD2 through mTOR regulation of FANCD2 gene transcripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation of ATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2 checkpoint activation by sustaining FANCD2. In FANCD2 deficient lymphoblasts, FANCD2 is essential to suppress endogenous and induced DNA damage, and FANCD2-deficient cells demonstrated impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by re-introduction of wild type FANCD2. Pharmacological inhibition of PI3K-mTOR-AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM, accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote the repair of DNA double strand breaks by sustaining FANCD2 and provide a novel mechanism of how the FA pathway modulates DNA damage response and repair.

The bromodomain and extra terminal domain (BET) inhibitor, JQ1 has marked antitumor activity against several hematologic malignancies as well as solid tumor models. Here we investigated its activity in vitro and in vivo against models of childhood rhabdomyosarcoma and Ewing sarcoma. In vitro, JQ1 (but not the inactive enantiomer JQ1R) inhibited cell proliferation, and increased G1 fraction of cells, although there was no correlation between cell line sensitivity and suppression of c-MYC or MYCN. In vivo, xenografts showed significant inhibition of growth during the period of treatment, and rapid regrowth after treatment was stopped, activity typical of antiangiogenic agents. Further, xenografts derived from cell lines intrinsically resistant or sensitive to JQ1 in vitro had similar sensitivity in vivo as xenografts. Further investigation showed that JQ1 reduced tumor vascularization. This was secondary to both drug-induced down regulation of tumor-derived growth factors and direct effects of JQ1 on vascular elements. JQ1 suppressed VEGF-stimulated vascularization of Matrigel plugs in mice, and in vitro suppressed differentiation, proliferation and invasion of human umbilical cord vascular endothelial cells (HUVECs). In HUVECs JQ1 partially suppressed c-MYC levels, but dramatically reduced AP-1 levels and activity through suppression of the AP-1 associated protein FOSL1. Our data suggest that the antitumor activity of JQ1 in these sarcoma models is largely a consequence of its anti-angiogenic activity.

Background-AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.

Previously, we reported that a predominant action of an IGF-1R-targeted antibody was through inhibiting tumor-derived VEGF, and indirectly, angiogenesis. Here we examined the direct anti-angiogenic activity of the IGF-1R-targeted antibody SCH717454 that inhibits ligand-receptor binding, and the mechanism by which tumors circumvent its anti-angiogenic activity. Inhibition of ligand stimulated activation of IGF-1R, insulin receptor (IN-R) or downstream signaling (phosphorylation of Akt [Ser473]) was determined by receptor-specific immunoprecipitation and immunoblotting. Inhibition of angiogenesis was determined by proliferation and tube formation using human umbilical vein endothelial cells (HUVECs) in vitro and in Matrigel plugs implanted in mice. SCH717454 blocked IGF-1 but not IGF-2 stimulated phosphorylation of Akt in sarcoma cells. Immunoprecipitation using anti-IGF-1R and anti-IN-R antibodies revealed that SCH717454 equally blocked IGF-1 and IGF-2 stimulated IGF-1R phosphorylation, but not IGF-2 stimulated phosphorylation of IN-R. SCH717454 completely blocked VEGF-stimulated proliferation and tube formation of HUVECs, but exogenous IGF-2 and insulin circumvented these inhibitory effects. Co-culture of HUVECs with IGF-2-secreting tumor cells completely abrogated SCH717454 inhibition of VEGF-stimulated HUVEC tube formation. In mice SCH717454 inhibited angiogenesis in VEGF-infused Matrigel plugs, but had no inhibitory activity when plugs contained both VEGF+IGF-2. These results reveal for the first time, a role for IGF-1R signaling in VEGF-mediated angiogenesis in vitro and indicate direct anti-angiogenic activity of SCH717454. Both in vitro and in vivo IGF-2 circumvented these effects through IN-R signaling. Many childhood cancers secrete IGF-2, suggesting that tumor-derived IGF-2 in the microenvironment maintains angiogenesis in the presence of IGF-1R-targeted antibodies allowing tumor progression.

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.