Peter J. Houghton

Treatment with the nucleoside analog cytarabine has been shown to mimic changes in gene expression associated with downregulation of the EWS‐FLI1 oncogene in Ewing sarcoma cell lines, selectively inhibit their growth in vitro, and cause tumor regression in athymic nude mice. For this report cytarabine was studied in vitro against a panel of 23 pediatric cancer cell lines and in vivo against 6 Ewing sarcoma xenografts. Acute lymphoblastic leukemia cell lines were the most sensitive to cytarabine in vitro (median IC₅₀ 9 nM), while Ewing sarcoma cell lines showed intermediate sensitivity (median IC₅₀ 232 nM). Cytarabine at a dose of 150 mg/kg administered daily 5× failed to significantly inhibit growth of five xenograft models, but reduced growth rate of the A673 xenograft by 50%. Cytarabine shows no differential in vitro activity against Ewing sarcoma cell lines and is ineffective in vivo against Ewing sarcoma xenografts at the dose and schedule studied. Pediatr Blood Cancer. 2010;55:1224–1226.

Background: Many childhood malignancies including sarcomas, neuroblastoma, and Wilms tumor show the presence of both, active, type‐1‐insulin‐like growth factor receptor (IGF‐1R), and the autocrine production of its ligands IGF‐1/IGF‐2. IMC‐A12 is a fully human IgG1 antibody that prevents ligand binding to the IGF‐1R.Procedures: IMC‐A12 was evaluated against the 23 cell lines of the Pediatric Preclinical Testing Program (PPTP) in vitro panel using 96 hr exposure at concentrations ranging from 0.01 nM to 0.1 µM. IMC‐A12 was tested in vivo at a dose of 1 mg/mouse administered intraperitoneally twice weekly for 6 weeks.Results: In vitro, IMC‐A12‐induced T/C values <50% in only three cell lines, a rhabdomyosarcoma cell line (Rh41) and two Ewing sarcoma cell lines (TC‐71 and CHLA‐9). In vivo, IMC‐A12 induced significant differences in EFS distribution compared to control in 24 of 34 (71%) evaluable solid tumor xenografts. Using the PPTP “time to event” activity measure, IMC‐A12 induced intermediate (n = 13) or high (n = 1) activity in 33 xenografts evaluable for this activity measure, including 6 of 6 rhabdomyosarcoma xenografts, 3 of 5 osteosarcoma xenografts, 2 of 5 neuroblastoma xenografts, and 1 of 5 Ewing sarcoma xenografts. The only objective response observed was observed in a rhabdomyosarcoma xenograft (Rh28) that achieved a maintained complete response.Conclusions: IMC‐A12 demonstrated broad antitumor activity against the PPTP's in vivo solid tumor panels, with the activity primarily being tumor growth inhibition rather than tumor regression. IMC‐A12 showed its greatest activity in vivo against the PPTP's rhabdomyosarcoma xenografts. Pediatr Blood Cancer 2010;54:921–926

Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S(473)), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.

Background: Ispinesib is a highly specific inhibitor of kinesin spindle protein (KSP, HsEg5), a mitotic kinesin required for separation of the spindle poles. Here we report the activity of ispinesib against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP).Procedures: Ispinesib was tested against the PPTP in vitro panel cell lines at concentrations from 0.1 nM to 1 µM and against the in vivo tumor panel xenografts by intraperitoneal administration (5 or 10 mg/kg) every 4 days for 3 doses repeated at day 21.Results: Ispinesib was highly potent against the PPTP's in vitro cell lines with a median IC₅₀ of 4.1 nM. Ispinesib (10 mg/kg) induced unexplained toxicity in mice bearing osteosarcoma xenografts and exceeded the MTD in 12 of 40 non‐osteosarcoma xenografts. Ispinesib induced significant tumor growth delay in 88% (23/26) of evaluable xenografts. Using a time to event measure of efficacy, ispinesib had intermediate and high levels of activity against 4 (21%) and 5 (26%) of the 19 evaluable solid tumor xenografts, respectively. Ispinesib induced maintained complete responses (CR) in a rhabdoid tumor, a Wilms tumor and a Ewing sarcoma xenograft. Ispinesib (5 mg/kg) produced 2 complete and 2 partial responses among 6 evaluable xenografts in the ALL panel. The in vivo pattern of activity was distinctive from that previously reported for vincristine.Conclusions: Ispinesib demonstrated broad in vivo antitumor activity, including maintained complete responses for several xenografts, although with high toxicity rates at the doses studied. Pediatr Blood Cancer 2009; 53:1255–1263.

Aplidin was tested in vitro at concentrations ranging from from 0.1 nM to 1.0 µM and in vivo at a dose of 0.6 mg/kg administered intraperitoneally on an every 4 days × 3‐schedule that was repeated at day 21. In vitro, Aplidin was most active against acute lymphoblastic leukemia (ALL) cell lines. In vivo, Aplidin induced significant differences in EFS distribution in 12 of 28 (43%) solid tumor models and 2 of 6 evaluable ALL models. Aplidin showed potent in vitro activity and induced significant in vivo tumor growth inhibition in some xenografts, but did not induce tumor regressions. Pediatr Blood Cancer 2009;53:509–512.

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.

Background 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. Procedures Alvespimycin was tested against the in vitro panel of the Pediatric Preclinical Testing Program (PPTP) at concentrations from 1 nM to 10 µM and was tested against the PPTP's in vivo tumor panels by intraperitoneal administration using a 50 mg/kg BID twice weekly × 6 weeks dose and schedule. Hsp70 induction in tumor and liver tissue was used as a pharmacodynamic measure of Hsp90 inhibition and stress response induction. Results Alvespimycin had a median IC₅₀ of 68 nM against the PPTP's in vitro panel, with a trend for lower IC₅₀ values for the rhabdomyosarcoma panel (median IC₅₀ 32 nM) and for higher IC₅₀ values for the neuroblastoma panel (median IC₅₀ 380 nM). Using the time to event activity measure, alvespimycin had intermediate or high activity against 4 of 28 evaluable solid tumor xenografts, including 3 of 4 alveolar rhabdomyosarcoma xenografts (one with a partial response). Hsp70 induction was observed in tumor tissue from both responding and non-responding xenografts. Conclusions Alvespimycin demonstrated little in vivo antitumor activity against most of the PPTP's preclinical models. The greatest drug effect was observed for the alveolar rhabdomyosarcoma xenografts in the rhabdomyosarcoma panel. Hsp70 induction was observed in responding and non-responding xenografts, suggesting that tumor-specific events subsequent to HSP90 inhibition are primary determinants of antitumor activity. Pediatr Blood Cancer 2008;51:34–41.