Doris A. Phelps

Nucleoside transport in normal and neoplastic cells

The permeation of nucleosides across the plasma membrane of mammalian cells is complex and mediated by at least five distinct transporters that differ in their sensitivity to inhibitors and in their specificity for nucleosides. The basic properties and permeant specificity of these transporters are summarized in Table 3. It appears that there may be differences in the distribution of these transporters in tumors and normal tissues that might be exploited for chemotherapeutic purposes. The human tumor cell lines examined express predominantly the NBMPR-sensitive equilibrative transporter es which can be blocked by low concentrations of NBMPR and dipyridamole. It is reasonable to expect that tumors with transport properties similar to the CCRF-CEM and Rh28 cell lines (Table 1) that have no detectable NBMPR-insensitive transport activity will be highly susceptible to the therapeutic approach of combining a transport inhibitor such as dipyridamole or NBMPR with an inhibitor of de novo pyrimidine biosynthesis. On the other hand, this approach to therapy is unlikely to succeed against tumors with transport phenotypes similar to the WI-L2 cell line that may permit the salvage nucleosides in the presence of these inhibitors. The majority of tumor cells examined, however, fall between these extremes, and it is not yet known what level of NBMPR-insensitive transport activity can be tolerated without seriously compromising this therapeutic approach. With respect to normal tissues, the mature absorptive cells of the intestine have predominantly Na(+)-dependent nucleoside transporters that are insensitive to NBMPR and dipyridamole. The proliferating crypt cells also appear to have Na(+)-dependent nucleoside transport, although they may also have an NBMPR-sensitive component of transport (Belt, unpublished data). Bone marrow granulocyte-macrophage progenitor cells also appear to have one or more concentrative nucleoside transporters. Thus these tissues, which are most vulnerable to the toxicity of antimetabolites, may be able to salvage nucleosides in the presence of inhibitors of equilibrative transport and be protected from the toxicity of de novo synthesis inhibitors. It is likely, however, that a successful application of this therapeutic approach will require the analysis of the nucleoside transport phenotype of individual tumors in order to identify those patients that may benefit from such therapy. Since the development of antibodies and cDNA probes for the various nucleoside transporters is currently underway in several laboratories, it is likely that analysis of the nucleoside transport phenotype of tumors from biopsy material will be feasible in the future.

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

Background Patients 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 Findings We 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). Conclusions The 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.

Molecular Characterization of the Pediatric Preclinical Testing Panel

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 Ewings 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.

Initial testing (stage 1) of AZD6244 (ARRY‐142886) by the pediatric preclinical testing program

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

Potent Inhibition of Angiogenesis by the IGF-1 Receptor-Targeting Antibody SCH717454 Is Reversed by IGF-2

Previously, we reported that a predominant action of a type-1 insulin-like growth factor receptor (IGF-1R)-targeted antibody was through inhibiting tumor-derived VEGF, and indirectly, angiogenesis. Here, we examined the direct antiangiogenic activity of the IGF-1R-targeted antibody SCH717454 that inhibits ligand-receptor binding and the mechanism by which tumors circumvent its antiangiogenic 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 (HUVEC) in vitro and in Matrigel plugs implanted in mice. SCH717454 blocked IGF-1-stimulated 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-stimulated 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. Coculture 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 antiangiogenic 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. Mol Cancer Ther; 11(3); 649–59. ©2011 AACR.

Regulation of FANCD2 by the mTOR pathway contributes to the resistance of cancer cells to DNA double strand breaks

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 to radiotherapy and sensitized rhabdomyosarcoma cells to the DNA interstrand cross-linker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia 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 showed impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by reintroduction of wild-type FANCD2. Pharmacologic 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 Fanconi anemia pathway modulates DNA damage response and repair.

Initial testing (stage 1) of lapatinib by the pediatric preclinical testing program.

Lapatinib is a small molecule reversible tyrosine kinase inhibitor of EGFR and ErbB2 that shows in vitro and in vivo activity against a range of EGFR and ErbB2‐dependent adult cancer cell lines and that has clinical efficacy against ErbB2‐overexpressing breast cancer.

Initial testing (stage 1) of the mTOR kinase inhibitor AZD8055 by the pediatric preclinical testing program.

Background AZD8055 is a small molecule ATP‐competitive inhibitor of the serine/threonine kinase mTOR that regulates cap‐dependent translation through the mTORC1 complex and Akt activation through the mTORC2 complex.

Stage 1 testing and pharmacodynamic evaluation of the HSP90 inhibitor alvespimycin (17-DMAG, KOS-1022) by the pediatric preclinical testing program

Alvespimycin (17‐DMAG, KOS‐1022), a potent small‐molecule inhibitor of the protein chaperone Hsp90, is being developed as an anticancer agent because of the multiple Hsp90 client proteins involved in cancer cell growth and survival.

Development, Characterization, and Reversal of Acquired Resistance to the MEK1 Inhibitor Selumetinib (AZD6244) in an In Vivo Model of Childhood Astrocytoma

Purpose: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAFV600E and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance. Experimental Design: BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD–resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed. Results: Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAFV600E astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40–resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD–resistant xenografts. Conclusions: Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance. Clin Cancer Res; 19(24); 6716–29. ©2013 AACR.