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1.
Nat Commun ; 14(1): 8373, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-38102140

ABSTRACT

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.


Subject(s)
Rhabdomyosarcoma , Humans , Carcinogenesis/genetics , Cell Line, Tumor , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Transcription Factors , Cell Transformation, Neoplastic , Cell Differentiation
2.
Mol Cancer Ther ; 22(4): 539-550, 2023 04 03.
Article in English | MEDLINE | ID: mdl-36696581

ABSTRACT

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.


Subject(s)
Nuclear Proteins , Sarcoma , Child , Humans , Transcription Factors , Receptor, IGF Type 1 , Sarcoma/drug therapy , Receptors, Somatomedin , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Cell Cycle Proteins , SOXF Transcription Factors
3.
Cell Oncol (Dordr) ; 45(1): 19-40, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34997546

ABSTRACT

BACKGROUND: The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown. METHODS: We used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining. RESULTS: We defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism. CONCLUSIONS: We show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis.


Subject(s)
Sarcoma, Ewing , Animals , Carcinogenesis , Cell Line, Tumor , Down-Regulation/genetics , Humans , Mice , Oncogene Proteins, Fusion/genetics , RNA , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology
4.
Cancers (Basel) ; 13(18)2021 Sep 09.
Article in English | MEDLINE | ID: mdl-34572755

ABSTRACT

Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.

5.
Pediatr Blood Cancer ; 67(7): e28284, 2020 07.
Article in English | MEDLINE | ID: mdl-32333633

ABSTRACT

BACKGROUND: VTP-50469 is a potent inhibitor of the menin-MLL1 interaction and is implicated in signaling downstream of EWSR1-FLI1. PROCEDURE: VTP-50469 was evaluated against seven Ewing sarcoma (EwS) xenograft models and in vitro against EwS cell lines. RESULTS: VTP-50469 showed limited antitumor activity, statistically significantly slowing tumor progression in four tumor models but with no evidence of tumor regression. In vitro, the IC50 concentration was 10 nM for the mixed lineage leukemia (MLL)-rearranged leukemia cell line MV4;11, but > 3 µM for EwS cell lines. CONCLUSIONS: In contrast to its high level of activity against MLL1-rearranged leukemia xenografts, VTP-50469 shows little activity against EwS models.


Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/drug therapy , Histone-Lysine N-Methyltransferase/drug effects , Myeloid-Lymphoid Leukemia Protein/drug effects , Proto-Oncogene Proteins/drug effects , Sarcoma, Ewing/drug therapy , Animals , Antineoplastic Agents/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Drug Evaluation, Preclinical , Female , Histone-Lysine N-Methyltransferase/metabolism , Humans , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Pediatrics , Proto-Oncogene Proteins/metabolism , Sarcoma, Ewing/pathology , Xenograft Model Antitumor Assays
6.
Clin Cancer Res ; 26(12): 3012-3023, 2020 06 15.
Article in English | MEDLINE | ID: mdl-32184294

ABSTRACT

PURPOSE: Vincristine combined with camptothecin derivatives showed synergy in preclinical pediatric cancer models, and the combinations are effective in treatment of childhood solid tumors. We determined whether the synergy between vincristine and irinotecan extends to eribulin, another microtubule inhibitor. EXPERIMENTAL DESIGN: Vincristine or eribulin, alone or combined with irinotecan, was studied in 12 xenograft models. Tumor regression and time to event were used to assess antitumor activity. Pharmacodynamic studies and RNA sequencing (RNA-seq) were conducted 24 and 144 hours after single-agent or combination treatment. Effects on vascular development were studied in Matrigel plugs implanted in mice. The interaction between binary combinations was examined in vitro. RESULTS: Eribulin combined with irinotecan was more effective than vincristine-irinotecan in 6 of 12 models. Pharmacodynamic markers induced by eribulin (phospho-histone H3) and irinotecan (γ-H2A.X) were abrogated in combination-treated tumors. The predominant RNA-seq signature in combination-treated tumors was activation of the TP53 pathway with increased nuclear TP53. Massive apoptosis was observed 24 hours only after treatment with the eribulin combination. In vitro, neither combination showed interaction using combination index analysis. Eribulin alone and the combination caused alterations in developing vasculature. CONCLUSIONS: The eribulin combination is very active in these xenograft models, but not synergistic in vitro. The combination reduced pharmacodynamic markers indicative of single-agent mechanisms but in tumors, dramatically activated the TP53 pathway. Although a mechanism for in vivo synergy requires further study, it is possible that eribulin-induced inhibition of microtubule dynamics enhances irinotecan-induced nuclear accumulation of TP53, leading to rapid cell death.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Kidney Neoplasms/drug therapy , Rhabdomyosarcoma/drug therapy , Wilms Tumor/drug therapy , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Female , Furans/administration & dosage , Gene Expression Profiling , Humans , Irinotecan/administration & dosage , Ketones/administration & dosage , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Mice , Mice, SCID , Prognosis , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/pathology , Tumor Cells, Cultured , Vincristine/administration & dosage , Wilms Tumor/genetics , Wilms Tumor/metabolism , Wilms Tumor/pathology , Xenograft Model Antitumor Assays
7.
Pediatr Blood Cancer ; 66(8): e27820, 2019 08.
Article in English | MEDLINE | ID: mdl-31099166

ABSTRACT

BACKGROUND: Entinostat, a selective class I histone deacetylase inhibitor, has been reported to enhance the activity of cytotoxic agents and suppress expression of PAX3-FOXO1 in alveolar rhabdomyosarcoma (ARMS). PROCEDURES: Entinostat was tested against three rhabdomyosarcoma cell lines using 96-hour drug exposure. Entinostat alone or in binary combination with vincristine, actinomycin D or cyclophosphamide was tested in ARMS and two embryonal rhabdomyosarcoma (ERMS) xenograft models. Tumor growth was measured at weekly intervals. Drug-induced changes in acetylated histone H3(K9) and entinostat pharmacokinetics were determined. RESULTS: In vitro, the IC50 concentration of entinostat ranged from 280 to 1300 nM. In vivo, entinostat significantly inhibited the growth of only Rh10 xenografts. For most studies, entinostat did not potentiate the activity of the cytotoxic agent. Exceptions included the vincristine and entinostat combination for Rh10 and the entinostat and actinomycin D combination for Rh10 and Rh18, although the effects were modest. For Rh18, the combination of entinostat with vincristine showed evidence of an antagonistic interaction compared with single-agent vincristine. Pharmacokinetic studies showed the average Cmax was 569.4 ng/mL (1.51 µM) with Tmax at 15 minutes, and total exposure (AUC0-12 h ) was 435.6 h × ng/mL. Entinostat treatment increased acetylated histone H3. CONCLUSIONS: Entinostat demonstrated modest antitumor activity in only one of four models at dose and shedule that gave drug exposures relevant to human treatment. The addition of entinostat to standard-of-care cytotoxic agents was in most instances no more effective than the cytotoxic agents used alone. Entinostat demonstrated target inhibition with increased histone 2A acetylation.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Cell Proliferation/drug effects , Standard of Care , Animals , Benzamides/administration & dosage , Breast Neoplasms/pathology , Cyclophosphamide/administration & dosage , Dactinomycin/administration & dosage , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Pyridines/administration & dosage , Tumor Cells, Cultured , Vincristine/administration & dosage , Xenograft Model Antitumor Assays
8.
JCI Insight ; 3(16)2018 08 23.
Article in English | MEDLINE | ID: mdl-30135299

ABSTRACT

Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bone Neoplasms/pathology , Interleukin-6/metabolism , Interleukin-8/metabolism , Lung Neoplasms/pathology , Adolescent , Adult , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/drug therapy , Bone and Bones/pathology , Cell Line, Tumor , Cell Proliferation , Child , Cytokine Receptor gp130/antagonists & inhibitors , Cytokine Receptor gp130/metabolism , Drug Evaluation, Preclinical , Follow-Up Studies , Humans , Hydrazines/pharmacology , Hydrazines/therapeutic use , Lung/pathology , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Male , Mice , Osteosarcoma/drug therapy , Osteosarcoma/prevention & control , Osteosarcoma/secondary , Primary Cell Culture , Quinoxalines/pharmacology , Quinoxalines/therapeutic use , Receptors, Interleukin-8A/antagonists & inhibitors , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Xenograft Model Antitumor Assays , Young Adult
9.
Pediatr Blood Cancer ; 65(2)2018 Feb.
Article in English | MEDLINE | ID: mdl-29080385

ABSTRACT

BACKGROUND: Integrating molecularly targeted agents with cytotoxic drugs used in curative treatment of pediatric cancers is complex. An evaluation was undertaken with the ERBB3/Her3-specific antibody patritumab (P) either alone or with the ERBB1/epidermal growth factor receptor inhibitor erlotinib (E) in combination with standard cytotoxic agents, cisplatin, vincristine, and cyclophosphamide, in pediatric sarcoma xenograft models that express receptors and ligands targeted by these agents. PROCEDURES: Tumor models were selected based upon ERBB3 expression and phosphorylation, and ligand (heregulin) expression. Patritumab, E, or these agents combined was evaluated without or with concomitant cytotoxic agents using procedures developed by the Pediatric Preclinical Testing Program. RESULTS: Full doses of cytotoxic agents were tolerated when combined with P, whereas dose reductions of 25% (vincristine, cisplatin) or 50% (cyclophosphamide) were required when combined with P + E. Patritumab, E alone, or in combination did not significantly inhibit growth of any tumor model, except for Rh18 xenografts (E alone). Patritumab had no single-agent activity and marginally enhanced the activity of vincristine and cisplatin only in Ewing sarcoma ES-4. P + E did not increase the antitumor activity of vincristine or cisplatin, whereas dose-reduced cyclophosphamide was significantly less active than cyclophosphamide administered at its maximum tolerated dose when combined with P + E. CONCLUSIONS: P had no single-agent activity, although it marginally potentiated the activity of vincristine and cisplatin in one of three models studied. However, the addition of E necessitated dose reduction of each cytotoxic agent, abrogating the enhancement observed with P alone.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bone Neoplasms/drug therapy , Erlotinib Hydrochloride/pharmacology , Sarcoma, Ewing/drug therapy , Animals , Antibodies, Monoclonal, Humanized , Bone Neoplasms/metabolism , Broadly Neutralizing Antibodies , Cell Line, Tumor , Female , Humans , Mice , Mice, SCID , Receptor, ErbB-3/antagonists & inhibitors , Receptor, ErbB-3/metabolism , Sarcoma, Ewing/metabolism , Xenograft Model Antitumor Assays
10.
Mol Cancer Ther ; 15(5): 1018-28, 2016 05.
Article in English | MEDLINE | ID: mdl-26908627

ABSTRACT

The bromodomain and extra-terminal domain 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. Furthermore, 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 downregulation 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 (HUVEC). 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 antiangiogenic activity. Mol Cancer Ther; 15(5); 1018-28. ©2016 AACR.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Neovascularization, Pathologic/metabolism , Sarcoma/metabolism , Sarcoma/pathology , Triazoles/pharmacology , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Child , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Human Umbilical Vein Endothelial Cells , Humans , Mice , Neovascularization, Pathologic/drug therapy , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Sarcoma/drug therapy , Transcription Factor AP-1/metabolism , Xenograft Model Antitumor Assays
11.
Pediatr Blood Cancer ; 62(10): 1768-74, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25981859

ABSTRACT

PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.


Subject(s)
Astrocytoma/genetics , Astrocytoma/radiotherapy , Benzimidazoles/adverse effects , Mutation , Proto-Oncogene Proteins B-raf/genetics , Radiotherapy/adverse effects , Animals , Blotting, Western , Cell Line, Tumor , Female , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, SCID , Multiprotein Complexes/metabolism , Neoplasms, Experimental/genetics , Neoplasms, Experimental/radiotherapy , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor Assays
12.
Pediatr Blood Cancer ; 62(8): 1345-52, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25832557

ABSTRACT

BACKGROUND: Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma; however, local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, although approximately 5-10% have MDM2 amplification or overexpression. PROCEDURES: The MDM2 inhibitor, RG7388, was examined alone or in combination with XRT (20Gy given in 2 Gy daily fractions) to immune-deficient mice bearing Rh18 (embryonal) or a total of 30 Gy in 2 Gy fractions to mice bearing Rh30 (alveolar) rhabdomyosarcoma xenografts. RG7388 was administered by oral gavage using two schedules (daily ×5; schedule 1 or once weekly; schedule 2). TP53-responsive gene products (p21, PUMA, DDB2, and MIC1) as well as markers of apoptosis were analyzed. RESULTS: RG7388 showed no significant single agent antitumor activity. Twenty Grays XRT induced complete regressions (CR) of Rh18 with 100 percent tumor regrowth by week 7, but no tumor regrowth at 20 weeks when combined with RG7388. RG7388 enhanced time to recurrence combined with XRT in Rh30 xenografts compared to 30 Gy XRT alone. RG7388 did not enhance XRT-induced local skin toxicity. Combination treatments induced TP53 responsive genes more rapidly and to a greater magnitude than single agent treatments. CONCLUSIONS: RG7388 enhanced the activity of XRT in both rhabdomyosarcoma models without increasing local XRT-induced skin toxicity. Changes in TP53-responsive genes were consistent with the synergistic activity of RG7388 and XRT in the Rh18 model.


Subject(s)
Apoptosis/radiation effects , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Pyrrolidines/therapeutic use , Radiation-Sensitizing Agents/therapeutic use , Rhabdomyosarcoma/radiotherapy , para-Aminobenzoates/therapeutic use , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Child , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins/metabolism , Female , Growth Differentiation Factor 15/metabolism , Humans , Mice , Mice, Nude , Neoplasm Recurrence, Local/epidemiology , Proto-Oncogene Proteins c-mdm2/biosynthesis , Rhabdomyosarcoma/drug therapy , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays
13.
Clin Cancer Res ; 19(24): 6716-29, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24132923

ABSTRACT

PURPOSE: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) 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 BRAF(V600E) 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.


Subject(s)
Astrocytoma/drug therapy , Astrocytoma/genetics , Benzimidazoles/administration & dosage , MAP Kinase Kinase 1/genetics , Animals , Anthraquinones/pharmacology , Astrocytoma/pathology , Cell Line, Tumor , Child , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , MAP Kinase Kinase 1/antagonists & inhibitors , Mice , Proto-Oncogene Proteins B-raf/genetics , STAT3 Transcription Factor/genetics , Sulfonamides/pharmacology
14.
Cancer Res ; 73(11): 3393-401, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23633493

ABSTRACT

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.


Subject(s)
DNA Breaks, Double-Stranded , Fanconi Anemia Complementation Group D2 Protein/genetics , Rhabdomyosarcoma/genetics , TOR Serine-Threonine Kinases/genetics , Adolescent , Animals , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Child , Child, Preschool , DNA Repair , Fanconi Anemia Complementation Group D2 Protein/metabolism , Female , Heterografts , Humans , Mice , Mice, SCID , Phosphorylation , Rhabdomyosarcoma/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor Assays
15.
Pediatr Blood Cancer ; 59(7): 1266-74, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-22315240

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Evaluation, Preclinical , Neoplasms, Experimental/drug therapy , Pyridinium Compounds/therapeutic use , Animals , Antineoplastic Agents/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Cell Line, Tumor , Child , Cyclic N-Oxides , Disease-Free Survival , Dose-Response Relationship, Drug , Female , Humans , Indolizines , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Neoplasm Transplantation , Pyridinium Compounds/pharmacokinetics , Xenograft Model Antitumor Assays
16.
Pediatr Blood Cancer ; 58(2): 191-9, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21337679

ABSTRACT

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.


Subject(s)
Cell Proliferation/drug effects , Morpholines/therapeutic use , Neoplasms, Experimental/drug therapy , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Blotting, Western , Child , Drug Evaluation, Preclinical , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/mortality , Survival Rate , Tumor Cells, Cultured
17.
Mol Cancer Ther ; 11(3): 649-59, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22188815

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Blood Vessels/drug effects , Insulin-Like Growth Factor II/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Angiogenesis Inhibitors/immunology , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Base Sequence , Blood Vessels/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Female , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, SCID , Molecular Sequence Data , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/prevention & control , Neovascularization, Physiologic/drug effects , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/immunology , Receptor, IGF Type 1/metabolism , Receptor, Insulin/antagonists & inhibitors , Receptor, Insulin/immunology , Receptor, Insulin/metabolism , Sarcoma/blood supply , Sarcoma/pathology , Vascular Endothelial Growth Factor A/pharmacology
18.
Pediatr Blood Cancer ; 57(3): 443-53, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21744473

ABSTRACT

BACKGROUND: PR-104 is rapidly hydrolyzed to PR-104A in vivo, which is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and amine (PR-104M) to produce DNA interstrand cross-links. PR-104 activation can occur via hypoxia-dependent reductases and also independently of hypoxia by aldo-keto reductase (AKR) 1C3. PROCEDURES: PR-104A was tested against the PPTP in vitro panel (10 nM to 100 µM), and PR-104 in vivo using a weekly × 6 schedule at its maximum tolerated dose (MTD) of 550 mg/kg. Subsequently PR-104 was tested at 270 and 110 mg/kg. Pharmacokinetics for PR-104 and its metabolites were determined, as were levels of AKR1C3 RNA and protein in xenografts. RESULTS: In vitro, the leukemia models were most sensitive to PR-104A. In vivo, PR-104 induced objective responses at its MTD in 21/34 solid tumor models and maintained complete responses against 7/7 acute lymphoblastic leukemia (ALL) models. At 270 mg/kg and lower dose levels, PR-104 did not induce solid tumor regressions, suggesting a steep dose-response relationship. Pharmacokinetic analysis suggests higher systemic exposures to PR-104A and its metabolites in mice compared to those achievable in patients. Levels of AKR1C3 protein did not correlate with tumor responsiveness. CONCLUSIONS: As monotherapy, PR-104 demonstrated a high level of activity against both solid tumor and ALL models at its MTD, but the activity was almost completely lost at half the MTD dose for solid tumors. Pharmacokinetic data at the PR-104 MTD from human trials suggest that PR-104 metabolites may not reach the plasma exposures in children that were associated with high-level preclinical activity.


Subject(s)
Nitrogen Mustard Compounds/metabolism , Nitrogen Mustard Compounds/pharmacokinetics , Prodrugs/metabolism , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation/drug effects , Child , Drug Screening Assays, Antitumor , Female , Humans , Maximum Tolerated Dose , Mice , Neoplasm Transplantation , Nitrogen Mustard Compounds/blood , Treatment Outcome , Xenograft Model Antitumor Assays
19.
Pediatr Blood Cancer ; 55(4): 668-77, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20806365

ABSTRACT

BACKGROUND: AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development. PROCEDURES: AZD6244 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel (1 nM-10 microM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily 5 days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting. RESULTS: At the highest concentration used in vitro (10 microM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild-type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment. CONCLUSIONS: At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.


Subject(s)
Benzimidazoles/therapeutic use , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neoplasms/drug therapy , Animals , Child , Drug Screening Assays, Antitumor , Female , Humans , Mice , Mice, Inbred BALB C , Mutation , Proto-Oncogene Proteins B-raf/genetics , Xenograft Model Antitumor Assays
20.
Pediatr Blood Cancer ; 53(4): 594-8, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19554571

ABSTRACT

BACKGROUND: 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. METHODS: Lapatinib was tested against the cell lines of the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10.0 microM. Lapatinib was tested against the xenografts of the PPTP in vivo panels using a twice-daily oral administration schedule for 6 weeks (5 days on, 2 days off) at a dose of 160 mg/kg (320 mg/kg/day). Lapatinib pharmacokinetic parameters were determined in scid(-/-) mice. RESULTS: The median IC(50) value for lapatinib against the entire PPTP cell line panel was 6.84 microM (range, 2.08 to >10.0 microM). Lapatinib was well tolerated in vivo, with toxicity in only 1.5% of the treated animals. Lapatinib induced significant differences in EFS distribution compared to controls in 1 of 41 xenografts tested. No objective responses were observed in any of the solid tumor panels or in the ALL panel. Lapatinib systemic exposure was consistent with previously observed values. CONCLUSIONS: Lapatinib has little activity against the xenografts of the PPTP's in vivo panel, and its in vitro activity occurs at concentrations above those associated with specific EGFR/ErbB2 inhibition. These results likely reflect lack of ErbB2 overexpression in the models studied and suggest that adult and pediatric cancers may fundamentally differ in the applicability of EGFR family members as therapeutic targets.


Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Animals , Cell Line, Tumor , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , Female , Genes, erbB-2 , Humans , Lapatinib , Mice , Quinazolines/pharmacokinetics , Receptor, ErbB-2/antagonists & inhibitors , Xenograft Model Antitumor Assays
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