Broad Spectrum Activity of the Checkpoint Kinase 1 Inhibitor Prexasertib as a Single Agent or Chemopotentiator Across a Range of Preclinical Pediatric Tumor Models.

PURPOSE: Checkpoint kinase 1 (CHK1) inhibitors potentiate the DNA-damaging effects of cytotoxic therapies and/or promote elevated levels of replication stress, leading to tumor cell death. Prexasertib (LY2606368) is a CHK1 small-molecule inhibitor under clinical evaluation in multiple adult and pediatric cancers. In this study, prexasertib was tested in a large panel of preclinical models of pediatric solid malignancies alone or in combination with chemotherapy. EXPERIMENTAL DESIGN: DNA damage and changes in cell signaling following in vitro prexasertib treatment in pediatric sarcoma cell lines were analyzed by Western blot and high content imaging. Antitumor activity of prexasertib as a single agent or in combination with different chemotherapies was explored in cell line-derived (CDX) and patient-derived xenograft (PDX) mouse models representing nine different pediatric cancer histologies. RESULTS: Pediatric sarcoma cell lines were highly sensitive to prexasertib treatment in vitro, resulting in activation of the DNA damage response. Two PDX models of desmoplastic small round cell tumor and one malignant rhabdoid tumor CDX model responded to prexasertib with complete regression. Prexasertib monotherapy also elicited robust responses in mouse models of rhabdomyosarcoma. Concurrent administration with chemotherapy was sufficient to overcome innate resistance or prevent acquired resistance to prexasertib in preclinical models of neuroblastoma, osteosarcoma, and Ewing sarcoma, or alveolar rhabdomyosarcoma, respectively. CONCLUSIONS: Prexasertib has significant antitumor effects as a monotherapy or in combination with chemotherapy in multiple preclinical models of pediatric cancer. These findings support further investigation of prexasertib in pediatric malignancies.

Initial Testing (Stage 1) of MK-8242-A Novel MDM2 Inhibitor-by the Pediatric Preclinical Testing Program.

BACKGROUND: MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction. PROCEDURES: MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 µM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models). RESULTS: The median IC50 for MK-8242 was 0.07 µM for TP53 wild-type cell lines versus >10 µM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials. CONCLUSIONS: MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.

Initial testing (stage 1) of the PARP inhibitor BMN 673 by the pediatric preclinical testing program: PALB2 mutation predicts exceptional in vivo response to BMN 673.

BACKGROUND: BMN 673 is a potent inhibitor of poly-ADP ribose polymerase (PARP) that is in clinical testing with a primary focus on BRCA-mutated cancers. BMN 673 is active both through inhibiting PARP catalytic activity and by tightly trapping PARP to DNA at sites of single strand breaks. PROCEDURE: BMN 673 was tested in vitro at concentrations ranging from 0.1 nM to 1 µM and in vivo at a daily dose of 0.33 mg/kg administered orally twice daily (Mon-Fri) and once daily on weekends (solid tumors) for 28 days. RESULTS: The median relative IC50 (rIC50 ) concentration against the PPTP cell lines was 25.8 nM. The median rIC50 for the Ewing cell lines was lower than for the remaining cell lines (6.4 vs. 31.1 nM, respectively). In vivo BMN 673 induced statistically significant differences in EFS distribution in 17/43 (39.5%) xenograft models. Three objective regressions were observed: a complete response (CR) in a medulloblastoma line (BT-45), a maintained CR in a Wilms tumor line (KT-10), and a maintained CR in an ependymoma line (BT-41). BMN 673 maintained its high level of activity against KT-10 with a threefold reduction in dose. KT-10 possesses a truncating mutation in PALB2 analogous to PALB2 mutations associated with hereditary breast and ovarian cancer that abrogate homologous recombination (HR) repair. CONCLUSIONS: The PPTP results suggest that single agent BMN 673 may have limited clinical activity against pediatric cancers. Single agent activity is more likely for patients whose tumors have defects in HR repair.

Initial testing (stage 1) of the topoisomerase II inhibitor pixantrone, by the pediatric preclinical testing program.

Pixantrone, a novel aza-anthracenedione with cytotoxic activity, was tested against the PPTP in vitro panel (3.0 nM to 30.0 µM) and against a limited panel of PPTP Wilms tumors and sarcomas (7.5 mg/kg) administered intravenously using an every 4 day × 3 schedule. In vitro pixantrone showed a median relative IC50 value of 54 nM (range <3 nM to 1.03 µM). In vivo pixantrone induced significant differences in EFS distribution compared to controls in two of eight solid tumor xenografts at dose levels relevant to human drug exposure. A complete response was observed for one Wilms tumor xenograft.

Initial testing (stage 1) of the histone deacetylase inhibitor, quisinostat (JNJ-26481585), by the Pediatric Preclinical Testing Program.

BACKGROUND: Quisinostat (JNJ-26481585) is a second-generation pyrimidyl-hydroxamic acid histone deacetylase (HDAC) inhibitor with high cellular potency towards Class I and II HDACs. Quisinostat was selected for clinical development as it showed prolonged pharmacodynamic effects in vivo and demonstrated improved single agent antitumoral efficacy compared to other analogs. PROCEDURES: Quisinostat was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 µM and was tested against the PPTP in vivo panels at a dose of 5 mg/kg (solid tumors) or 2.5 mg/kg (ALL models) administered intraperitoneally daily × 21. RESULTS: In vitro quisinostat demonstrated potent cytotoxic activity, with T/C% values approaching 0% for all of the cell lines at the highest concentration tested. The median relative IC50 value for the PPTP cell lines was 2.2 nM (range <1-19 nM). quisinostat induced significant differences in EFS distribution compared to control in 21 of 33 (64%) of the evaluable solid tumor xenografts and in 4 of 8 (50%) of the evaluable ALL xenografts. An objective response was observed in 1 of 33 solid tumor xenografts while for the ALL panel, two xenografts achieved complete response (CR) or maintained CR, and a third ALL xenograft achieved stable disease. CONCLUSIONS: Quisinostat demonstrated broad activity in vitro, and retarded growth in the majority of solid tumor xenografts studied. The most consistent in vivo activity signals observed were for the glioblastoma xenografts and T-cell ALL xenografts.

C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.

We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.

Initial testing of the MDM2 inhibitor RG7112 by the Pediatric Preclinical Testing Program.

BACKGROUND: RG7112 is a selective inhibitor of p53-MDM2 binding that frees p53 from negative control, activating the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. RG7112 was selected for evaluation by the Pediatric Preclinical Testing Program (PPTP) due to the relatively low incidence of p53 mutations in pediatric cancers compared with adult malignancies. PROCEDURES: RG7112 and its inactive enantiomer RG7112i were evaluated against the 23 cell lines of the PPTP in vitro panel using 96 hours exposure (1 nM to 10 µM). It was tested against the PPTP in vivo panel focusing on p53 wild-type (WT) xenografts at a dose of 100 mg/kg daily for 14 days followed by 4 weeks of observation. Response outcomes were related to MDM2 and p53 expression datasets (http://pptp.nchresearch.org/data.html). RESULTS: RG7112 demonstrated cytotoxic activity with a lower median IC(50) for p53 WT versus p53 mutant cell lines (approximately 0.4 µM vs. >10 µM, respectively). RG7112 induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C > 2) in 10 of 26 (38%) solid tumor xenografts. Objective responses included medulloblastoma, alveolar rhabdomyosarcoma, Wilms, rhabdoid and Ewing sarcoma xenografts. For the ALL panel, there was one partial response, five complete responses and one maintained complete response. The ALL xenografts expressed the highest levels of p53 among the PPTP panels. CONCLUSIONS: RG7112 induced tumor regressions in solid tumors from different histotype panels, and exhibited consistent high-level activity against ALL xenografts. This high level of activity supports prioritization of RG7112 for further evaluation.

Initial testing (stage 1) of the cyclin dependent kinase inhibitor SCH 727965 (dinaciclib) by the pediatric preclinical testing program.

BACKGROUND: SCH 727965 is a novel drug in clinical development that potently and selectively inhibits CDK1, CDK2, CDK5, and CDK9. The activity of SCH 727965 was evaluated against the PPTP's in vitro and in vivo panels. PROCEDURES: SCH 727965 was tested against the PPTP in vitro panel using 96 hours exposure at concentrations ranging from 0.1 nM to 1.0 µM. It was tested against the PPTP in vivo panels at a dose of 40 mg/kg administered intraperitoneally twice weekly for 2 weeks and repeated at Day 21 with a total observation period of 6 weeks. RESULTS: The median IC(50) value for the cell lines was 7.5 nM, with less than fourfold range between the minimum (3.4 nM) and maximum (11.2 nM) IC(50) values. SCH 727965 demonstrated an activity pattern consistent with cytotoxicity for most of the cell lines. Forty-three xenograft models were studied and SCH 727965 induced significant delays in event free survival distribution compared to control in 23 of 36 (64%) evaluable solid tumor xenografts and in 3 of 7 ALL xenografts. SCH 727965 did not induce objective responses in the solid tumor panels and the best response observed was stable disease for one osteosarcoma xenograft. In the leukemia panel, there were two objective responses with a complete response observed in a single xenograft. CONCLUSIONS: SCH 727965 shows an interesting pattern of activity suggesting its potential applicability against selected childhood cancers, particularly leukemias.

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. Procedures AZD8055 was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 µM and against the PPTP in vivo panels at a dose of 20 mg/kg administered orally daily x 7 for 4 weeks. RESULTS: In vitro the median relative IC(50) for AZD8055 against the PPTP cell lines was 24.7 nM. Relative I/O values >0% (consistent with a cytostatic effect) were observed in 8 cell lines and 15 cell lines showed Relative I/O values ranging from -4.7 to -92.2% (consistent with varying degrees of cytotoxic activity). In vivo AZD8055 induced significant differences in EFS distribution compared to controls in 23 of 36 (64%) evaluable solid tumor xenografts, and 1 of 6 evaluable ALL xenografts. Intermediate activity for the time to event activity measure (EFS T/C >2) was observed in 5 of 32 (16%) solid tumor xenografts evaluable. The best response was stable disease. PD2 (progressive disease with growth delay) was observed in 20 of 36 (55.6%) evaluable solid tumor xenografts. AZD8055 significantly inhibited 4E-BP1, S6, and Akt phosphorylation following day 1 and day 4 dosing, but suppression of mTORC1 or mTORC2 signaling did not predict tumor sensitivity. CONCLUSIONS: AZD8055 demonstrated broad activity in vitro, but at the dose and schedule studied demonstrated limited activity in vivo against the PPTP solid tumor and ALL panels.

Testing of the topoisomerase 1 inhibitor Genz-644282 by the pediatric preclinical testing program.

BACKGROUND: Genz-644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development. PROCEDURES: Genz-644282 was tested against the PPTP in vitro panel (0.1 nM to 1 µM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz-644282 was tested at 4, 3, 2, and 1 mg/kg in 3 models to assess the dose-response relationship. mRNA gene signatures predictive for Genz-644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively. RESULTS: In vitro, Genz-644282 demonstrated potent cytotoxic activity with a median IC(50) of 1.2 nM (range 0.2-21.9 nM). In vivo, Genz-644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz-644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz-644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz-644282. CONCLUSIONS: Genz-644282 was highly active within a narrow dose range (2-4 mg/kg), typical of other topoisomerase I poisons. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent.

Initial testing of JNJ-26854165 (Serdemetan) by the pediatric preclinical testing program.

JNJ-26854165 was originally developed as an activator of p53 capable of inducing apoptosis in cancer cell lines. In vitro, JNJ-26854165 demonstrated cytotoxic activity. The ALL cell line panel had a significantly lower median IC(50) (0.85 µM) than the remaining cell lines. In vivo JNJ-26854165 induced significant differences in EFS distribution compared to control in 18 of 37 solid tumors and in 5 of 7 of the evaluable ALL xenografts. Objective responses were observed in 4 of 37 solid tumor xenografts, and 2 of 7 ALL xenografts achieved PR or CR. Responses were noted in xenografts with both mutant and wild-type p53.

Initial testing (stage 1) by the pediatric preclinical testing program of RO4929097, a γ-secretase inhibitor targeting notch signaling.

RO4929097 is a potent and selective inhibitor of γ-secretase and as a result is able to inhibit Notch pathway signaling. The activity of RO4929097 was evaluated against the in vivo panels of the Pediatric Preclinical Testing Program (PPTP). RO4929097 induced significant differences in event-free survival (EFS) distribution compared to control in 6 of 26 (23%) of the evaluable solid tumor xenografts and in 0 of 8 (0%) of the evaluable ALL xenografts. The most consistent tumor growth delay effects were noted in the osteosarcoma panel. RO4929097 at the dose and schedule evaluated demonstrated little antitumor activity against childhood cancer xenografts.

Initial testing (stage 1) of the polyamine analog PG11047 by the pediatric preclinical testing program.

BACKGROUND: PG11047 is a novel conformationally restricted analog of the natural polyamine, spermine that lowers cellular endogenous polyamine levels and competitively inhibits natural polyamine functions leading to cancer cell growth inhibition. The activity of PG11047 was evaluated against the PPTP's in vitro and in vivo panels. PROCEDURES: PG11047 was evaluated against the PPTP in vitro panel using 96 hr exposure at concentrations ranging from 10 nM to 100 µM. It was tested against the PPTP in vivo panels at a dose of 100 mg/kg administered by the intraperitoneal route weekly for 6 weeks. RESULTS: In vitro PG11047 demonstrated a concentration-response pattern consistent with cytostatic activity. The median EC(50) for PG11047 was 71 nM. Cell lines of the Ewing sarcoma panel had a lower median EC(50) value compared to the remaining cell lines in the panel, while cell lines of the neuroblastoma panel had a higher median EC(50) value. In vivo PG11047 induced significant differences in EFS distribution compared to control in 5 of 32 (15.6%) of the evaluable solid tumor xenografts and in 0 of 7 (0%) of the evaluable ALL xenografts. The single case of tumor regression occurred in an ependymoma xenograft. CONCLUSIONS: Further pediatric development of PG11047 will require better defining a target population and identifying combinations for which there is a tumor-selective cytotoxic effect. The regression observed for an ependymoma xenograft and the exquisite sensitivity of some Ewing sarcoma cell lines to the antiproliferative effects of PG11047 provide leads for further preclinical investigations.

Initial testing (stage 1) of the multi-targeted kinase inhibitor sorafenib by the pediatric preclinical testing program.

BACKGROUND: Sorafenib is an inhibitor of multiple kinases (e.g., VEGF receptors, PDGFR, FLT3, RET, BRAF, KIT) and is approved by FDA for treatment of two adult cancers. The activity of sorafenib was evaluated against the PPTP's in vitro and in vivo panels. PROCEDURES: Sorafenib was evaluated against the PPTP in vitro panel using 96-hr exposure at concentrations ranging from 1.0 nM to 10.0 µM. It was tested against the PPTP in vivo panels at a dose of 60 mg/kg administered by oral gavage daily for 5 days per week, repeated for 6 weeks. RESULTS: In vitro sorafenib demonstrated cytotoxic activity, with a median IC(50) value of 4.3 µM. Twenty of 23 cell lines had IC(50) values between 1.0 and 10.0 µM. A single cell line (Kasumi-1) with an activating KIT mutation had an IC(50) value < 1.0 µM (IC(50) = 0.02 µM). In vivo sorafenib induced significant differences in event-free survival (EFS) distribution compared to control in 27 of 36 (75%) of the evaluable solid tumor xenografts and in 1 of 8 (12.5%) of the evaluable ALL xenografts. Sorafenib induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C) in 15 of 34 (44%) evaluable solid tumor xenografts. No xenografts achieved an objective response. CONCLUSIONS: The primary in vitro activity of sorafenib was noted at concentrations above 1 µM, with the exception of a more sensitive cell line with an activating KIT mutation. The primary in vivo effect for sorafenib was tumor growth inhibition, which was observed across multiple histotypes.

Initial testing (stage 1) of mapatumumab (HGS-ETR1) by the pediatric preclinical testing program.

Mapatumumab (HGS-ETR1) is a fully human IgG1 agonistic monoclonal antibody that exclusively targets and activates tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1). It was tested in vitro at concentrations from 0.01 to 100 microg/ml and in vivo at a dose of 10 mg/kg administered intraperitoneally using a twice-weekly schedule. Mapatumumab demonstrated limited activity against the 23 cell lines of the PPTP in vitro panel with no lines achieving 50% growth inhibition. Mapatumumab induced significant differences in event-free survival distribution compared to controls in 9 of 37 evaluable solid tumor xenografts tested, but in none of the 8 ALL xenografts.

Initial testing of topotecan by the pediatric preclinical testing program.

BACKGROUND: Topotecan is a small molecule DNA topoisomerase I poison, that has been successful in clinical trials against pediatric solid tumors and leukemias. Topotecan was evaluated against the Pediatric Preclinical Testing Program (PPTP) tumor panels as part of a validation process for these preclinical models. PROCEDURES: In vivo three measures of antitumor activity were used: (1) an objective response measure modeled after the clinical setting; (2) a treated to control (T/C) tumor volume measure; and (3) a time to event (fourfold increase in tumor volume for solid tumor models, or > or =25% human CD45+ cells in the peripheral blood for acute lymphoblastic leukemia, ALL models) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. RESULTS: Topotecan inhibited cell growth in vitro with IC(50) values between 0.71 and 489 nM. Topotecan significantly increased EFS in 32 of 37 (87%) solid tumor xenografts and in all 8 of the ALL xenografts. Seventy-five percent of solid tumors met EFS T/C activity criteria for intermediate (n = 17) or high activity (n = 7). Objective responses were noted in eight solid tumor xenografts (Wilms, rhabdomyosarcoma, Ewing sarcoma, neuroblastoma). Among the six neuroblastomas, three achieved a PR. For the ALL panel, two maintained CRs, three CRs, and two PRs were observed. CONCLUSIONS: Topotecan demonstrated broad activity in vitro and in vivo against both the solid tumor and ALL panels, with significant tumor growth delay generated in all the panels. These results further demonstrate the validity of the PPTP panel for preclinical testing of new drugs.