Abstract
Neuroblastoma is a pediatric cancer that is frequently metastatic and resistant to conventional treatment. In part, a lack of natively metastatic, chemoresistant in vivo models has limited our insight into the development of aggressive disease. The Th-MYCN genetically engineered mouse model develops rapidly progressive chemosensitive neuroblastoma and lacks clinically relevant metastases. To study tumor progression in a context more reflective of clinical therapy, we delivered multicycle treatment with cyclophosphamide to Th-MYCN mice, individualizing therapy using MRI, to generate the Th-MYCN CPM32 model. These mice developed chemoresistance and spontaneous bone marrow metastases. Tumors exhibited an altered immune microenvironment with increased stroma and tumor-associated fibroblasts. Analysis of copy number aberrations revealed genomic changes characteristic of human MYCN-amplified neuroblastoma, specifically copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. RNA sequencing revealed enriched expression of genes associated with 17q gain and upregulation of genes associated with high-risk neuroblastoma, such as the cell-cycle regulator cyclin B1-interacting protein 1 (Ccnb1ip1) and thymidine kinase (TK1). The antiapoptotic, prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, and treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival. Our results highlight that under treatment conditions that mimic chemotherapy in human patients, Th-MYCN mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease. The Th-MYCN CPM32 model therefore is a useful tool to dissect in detail mechanisms that drive metastasis and chemoresistance, and highlights dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease. SIGNIFICANCE: An in vivo mouse model of high-risk treatment-resistant neuroblastoma exhibits changes in the tumor microenvironment, widespread metastases, and sensitivity to JAK1/2 inhibition.
Highlights
Neuroblastoma is an aggressive tumor of neural crest origin
Using the clinical JAK1/JAK2 oral inhibitor CYT387, currently in phase III clinical trials in adult cancer (NCT02101021), we show that cell lines derived from cyclophosphamide-resistant tumors are sensitive to JAK1/2 inhibition and that in vivo treatment with CYT387 and cyclophosphamide significantly improved survival of Th-MYCN mice bearing cyclophosphamide-resistant tumors
To identify mechanisms associated with the development of chemoresistance in neuroblastoma, we developed an individualized and MRI-guided dose escalation schedule for cyclophosphamide treatment using the homozygous Th-MYCN model, in which 100% of homozygotes develop tumors with a mean onset of 35 Æ 7.5 days
Summary
Approximately half of patients have high-risk disease defined by the presence of metastatic disease, amplification of the MYCN oncogene or other clinical risk criteria. Conventional multimodal treatment is intensive and is characterized by frequent development of chemoresistant, metastatic disease [1, 2], reinforcing the need to develop mechanistically targeted treatments for chemorefractory patients. Chemoresistance relating to increased drug efflux via MDR1 and MRP transport for MRP/MDR drug substrates, and temozolomide resistance related to upregulation of MGMT activity is well characterized [3, 4]. Evidence for intratumoral clonal selection as a consequence of conventional treatment is emerging from sequencing of paired diagnostic:relapse tissue biopsies, where evolution of new mutations not present at diagnosis, and clonal enrichment of alterations in a limited number of genes such as HRAS, BRAF, and ALK is described [5,6,7].
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