Systematic target actionability reviews of preclinical proof-of-concept papers to match targeted drugs to paediatric cancers.

BACKGROUND: Children with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. METHODS: A sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for paediatric tumour type and categorised into nine separate PoC data modules. Each experimental finding was scored for experimental outcome and quality independently by two reviewers; discrepancies were assessed by a third reviewer and resolved by adjudication. Scores corresponding to one PoC module were merged for each tumour type and visualised in a heat map matrix in the publicly available R2 data portal [r2.amc.nl]. RESULTS AND CONCLUSIONS: To test our TAR methodology, we conducted a pilot study on MDM2 and TP53. The heat map generated from analysis of 161 publications provides a rationale to support drug development in specific paediatric solid and brain tumour types. Furthermore, our review highlights tumour types where preclinical data are incomplete or lacking and for which additional preclinical testing is advisable.

A pan-cancer transcriptome analysis identifies replication fork and innate immunity genes as modifiers of response to the CHK1 inhibitor prexasertib.

The combined influence of oncogenic drivers, genomic instability, and/or DNA damage repair deficiencies increases replication stress in cancer. Cells with high replication stress rely on the upregulation of checkpoints like those governed by CHK1 for survival. Previous studies of the CHK1 inhibitor prexasertib demonstrated activity across multiple cancer types. Therefore, we sought to (1) identify markers of prexasertib sensitivity and (2) define the molecular mechanism(s) of intrinsic and acquired resistance using preclinical models representing multiple tumor types. Our findings indicate that while cyclin E dysregulation is a driving mechanism of prexasertib response, biomarkers associated with this aberration lack sufficient predictive power to render them clinically actionable for patient selection. Transcriptome analysis of a pan-cancer cell line panel and in vivo models revealed an association between expression of E2F target genes and prexasertib sensitivity and identified innate immunity genes associated with prexasertib resistance. Functional RNAi studies supported a causal role of replication fork components as modulators of prexasertib response. Mechanisms that protect cells from oncogene-induced replication stress may safeguard tumors from such stress induced by a CHK1 inhibitor, resulting in acquired drug resistance. Furthermore, resistance to prexasertib may be shaped by innate immunity.

Investigating a chimeric anti-mouse PDGFRα antibody as a radiosensitizer in primary mouse sarcomas.

BACKGROUND: Olaratumab (LY3012207/IMC-3G3/Lartruvo™) is a fully human monoclonal antibody specific for platelet-derived growth factor receptor alpha (PDGFRα). Phase Ib/II trial results of olaratumab plus doxorubicin in adult patients with advanced soft tissue sarcoma (STS) supported accelerated FDA approval of this regimen. Radiation therapy (RT) is frequently used for high-risk localized STS. However, olaratumab has not been tested with concurrent RT. Here, we evaluate the chimeric anti-mouse PDGFRα antibody 1E10Fc as a radiosensitizer in a primary mouse model of STS. METHODS: Primary STS were initiated in mice. When tumors reached 70 mm3, mice were allocated into treatment groups: 1) isotype, 2) 1E10Fc, 3) isotype + RT, 4) 1E10Fc + RT. 1E10Fc or isotype was given biweekly. RT (25 Gy delivered in 5 daily 5 Gy fractions) was initiated on Day 0 with first drug treatment. Tumors were measured 3× per week. Upon reaching 900 mm3, tumors and lungs were harvested. A two-way ANOVA was performed to compare tumor growth delay. Primary tumors were stained for CD31 and PDGFRα and lungs were assessed for micrometastases. A Chi-square test was performed to compare the development of micrometastases in the lungs after treatment with 1E10Fc or isotype. FINDINGS: RT significantly delayed time to tumor quintupling compared to no RT (p < 0·0001) [two-way ANOVA], but no difference in tumor growth was seen between mice receiving isotype or 1E10Fc treatment regardless of concurrent RT. Lower microvessel density was observed in the 1E10Fc + RT group. Fewer mice treated with 1E10Fc had micrometastases, but this difference was not statistically significant (p < 0·09). INTERPRETATION: 1E10Fc did not act as a radiosensitizer in this primary STS model. FUNDING: This study was funded by a research agreement from Eli Lilly and Company.

Anti-VEGFR2 therapy delays growth of preclinical pediatric tumor models and enhances anti-tumor activity of chemotherapy.

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target in solid malignancies due to its central role in tumor angiogenesis. Ramucirumab (Cyramza®, LY3009806) is a human monoclonal antibody specific for VEGFR2 approved for several adult indications and currently in a phase 1 clinical trial for pediatric patients with solid tumors (NCT02564198). Here, we evaluated ramucirumab in vitro and the anti-murine VEGFR2 antibody DC101 in vivo with or without chemotherapy across a range of pediatric cancer models. Ramucirumab abrogated in vitro endothelial cord formation driven by cancer cell lines representing multiple pediatric histologies; this response was independent of the origin of the tumor cell-line. Several pediatric cancer mouse models responded to single agent DC101-mediated VEGFR2 inhibition with tumor growth delay. Preclinical stable disease and partial xenograft regressions were observed in mouse models of Ewing's sarcoma, synovial sarcoma, neuroblastoma, and desmoplastic small round cell tumor treated with DC101 and cytotoxic chemotherapy. In contrast, DC101 treatment in osteosarcoma models had limited efficacy alone or in combination with chemotherapeutics. Our data indicate differential efficacy of targeting the VEGFR2 pathway in pediatric models and support the continued evaluation of VEGFR2 inhibition in combination with cytotoxic chemotherapy in multiple pediatric indications.

MAGE-A3 is a Clinically Relevant Target in Undifferentiated Pleomorphic Sarcoma/Myxofibrosarcoma.

Melanoma-associated antigen 3 (MAGE-A3) expression is generally restricted to the placenta and germline cells of the testis, but it may also be expressed in sarcoma and other cancers and is associated with poor prognosis. Immunotherapy approaches targeting MAGE-A3 in other cancers have shown mixed results in the clinic, however, use of cancer testis antigens such as MAGE-A3 may have therapeutic value in the treatment of soft tissue sarcomas. Based on the recent success of anti-programmed death-1 (PD-1) therapy in undifferentiated pleomorphic sarcoma, we hypothesize that MAGE-A3-based immunotherapies may also provide benefits in this sarcoma type. We analyzed MAGE-A3 expression of sarcoma subtypes available in the Cancer Genome Atlas and Cancer Cell Line Encyclopedia and show that undifferentiated pleomorphic sarcoma/myxofibrosarcoma (UPS/MFS) expresses this potential target gene. We have identified high protein expression by tissue microarray of 106 UPS cores. We also found that high MAGE-A3 mRNA and protein expression is associated with worse overall survival in UPS/MFS. Furthermore, our results show no human leukocyte antigen (HLA) expression loss and relatively high lymphocyte infiltration by lymphocyte specific protein tyrosine kinase (LCK) marker expression. Based on these results, we propose targeting MAGE-A3 in UPS/MFS by immunotherapy techniques.

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.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α.

Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors.Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor.Results:In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin.Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847-57. ©2017 AACR.

Abemaciclib Is Active in Preclinical Models of Ewing Sarcoma via Multipronged Regulation of Cell Cycle, DNA Methylation, and Interferon Pathway Signaling.

PURPOSE: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. EXPERIMENTAL DESIGN: Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. RESULTS: Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. CONCLUSIONS: Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Three-Dimensional Patient-Derived In Vitro Sarcoma Models: Promising Tools for Improving Clinical Tumor Management.

Over the past decade, the development of new targeted therapeutics directed against specific molecular pathways involved in tumor cell proliferation and survival has allowed an essential improvement in carcinoma treatment. Unfortunately, the scenario is different for sarcomas, a group of malignant neoplasms originating from mesenchymal cells, for which the main therapeutic approach still consists in the combination of surgery, chemotherapy, and radiation therapy. The lack of innovative approaches in sarcoma treatment stems from the high degree of heterogeneity of this tumor type, with more that 70 different histopathological subtypes, and the limited knowledge of the molecular drivers of tumor development and progression. Currently, molecular therapies are available mainly for the treatment of gastrointestinal stromal tumor, a soft-tissue malignancy characterized by an activating mutation of the tyrosine kinase KIT. Since the first application of this approach, a strong effort has been made to understand sarcoma molecular alterations that can be potential targets for therapy. The low incidence combined with the high level of histopathological heterogeneity makes the development of clinical trials for sarcomas very challenging. For this reason, preclinical studies are needed to better understand tumor biology with the aim to develop new targeted therapeutics. Currently, these studies are mainly based on in vitro testing, since cell lines, and in particular patient-derived models, represent a reliable and easy to handle tool for investigation. In the present review, we summarize the most important models currently available in the field, focusing in particular on the three-dimensional spheroid/organoid model. This innovative approach for studying tumor biology better represents tissue architecture and cell-cell as well as cell-microenvironment crosstalk, which are fundamental steps for tumor cell proliferation and survival.

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2-M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR.