Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRAS Q61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations.Significance: These findings suggest that conventional therapyresistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

Osteosarcoma (OS) is the most common malignant bone tumor and the majority of recurrences are due to metastasis. However, the molecular mechanisms that regulate OS metastatic spread are largely unknown. In this study, we report that special AT-rich-binding protein 2 (SATB2) is highly expressed in OS cells and tumors. Short hairpin RNA-mediated knockdown of SATB2 (sh-SATB2) decreases migration and invasion of OS cells without affecting proliferation or viability. Microarray analysis identified genes that were differentially regulated by SATB2 including the actin-binding protein Epithelial Protein Lost In Neoplasm (EPLIN), which was upregulated in sh-SATB2 cells. Silencing EPLIN rescues the decreased invasion observed in sh-SATB2 cells. Pathway analyses of SATB2-regulated genes revealed enrichment of those involved in cytoskeleton dynamics, and increased stress fiber formation was detected in cells with SATB2 knockdown. Furthermore, sh-SATB2 cells exhibit increased RhoA, decreased Rac1 and increased phosphorylation of focal adhesion kinase (FAK) and paxillin. These findings identify SATB2 as a novel regulator of OS invasion, in part via effects on EPLIN and the cytoskeleton.

Metastatic relapse is the major cause of death in pediatric neuroblastoma, where there remains a lack of therapies to target this stage of disease. To understand the molecular mechanisms mediating neuroblastoma metastasis, we developed a mouse model using intracardiac injection and in vivo selection to isolate malignant cell subpopulations with a higher propensity for metastasis to bone and the central nervous system. Gene expression profiling revealed primary and metastatic cells as two distinct cell populations defined by differential expression of 412 genes and of multiple pathways, including CADM1, SPHK1, and YAP/TAZ, whose expression independently predicted survival. In the metastatic subpopulations, a gene signature was defined (MET-75) that predicted survival of neuroblastoma patients with metastatic disease. Mechanistic investigations demonstrated causal roles for CADM1, SPHK1, and YAP/TAZ in mediating metastatic phenotypes in vitro and in vivo Notably, pharmacologic targeting of SPHK1 or YAP/TAZ was sufficient to inhibit neuroblastoma metastasis in vivo Overall, we identify gene expression signatures and candidate therapeutics that could improve the treatment of metastatic neuroblastoma. Cancer Res; 77(3); 696-706. ©2017 AACR.

Highlights d ALK extracellular domain (ECD) cleavage occurs at Asn 654-Leu655 d ECD cleavage mediates neuroblastoma (NB) cell migration d Cleavage inhibition leads to downregulated nuclear b-catenin and EMT gene signatures d ECD cleavage is caused by MMP-9 whose inhibition decreases NB cell migration

Oral metronomic topotecan represents a novel approach to chemotherapy delivery which, in preclinical models, may work synergistically with pazopanib in targeting angiogenesis. A phase I and pharmacokinetic (PK) study of this combination was performed in children with relapsed/refractory solid tumors. Oral topotecan and pazopanib were each administered daily without interruption in 28-day cycles at five dose levels (0.12 to 0.3 mg/m2 topotecan and 125 to 160 mg/m2 pazopanib powder for oral suspension (PfOS)), with dose escalation in accordance with the rolling-six design. PK studies were performed on day 1 and at steady state. Thirty patients were enrolled, with 26 evaluable for dose-limiting toxicity (DLT), with median age 12 years (3–20). Toxicities were generally mild; the most common grade 3/4 adverse events related to protocol therapy were neutropenia (18%), thrombocytopenia (11%), lymphopenia (11%), AST elevation (11%), and lipase elevation (11%). Only two cycle 1 DLTs were observed on study, both at the 0.3/160 mg/m2 dose level comprising persistent grade 3 thrombocytopenia and grade 3 ALT elevation. No AEs experienced beyond cycle 1 required treatment discontinuation. The best response was stable disease in 10/25 patients (40%) for a median duration of 6.4 (1.7–45.1) months. The combination of oral metronomic topotecan and pazopanib is safe and tolerable in pediatric patients with solid tumors, with a recommended phase 2 dose of 0.22 mg/m2 topotecan and 160 mg/m2 pazopanib. No objective responses were observed in this heavily pre-treated patient population, although 40% did achieve stable disease for a median of 6 months. While this combination is likely of limited benefit for relapsed disease, it may play a role in the maintenance setting.

Survival for high-risk neuroblastoma remains poor. Most patients who recur, present with metastatic disease, and few targetable pathways that govern spread to distant sites are currently known. We previously developed a metastatic mouse model to select cells with enhanced ability to spread to the bone and brain and identified a signature based on differentially expressed genes, which also predicted patient survival. To discover new neuroblastoma therapies, we utilized the Connectivity Map to identify compounds that can reverse this metastatic transcriptional signature and found calcipotriol, a vitamin D3 analog, to be a compound that selectively targets cell lines with enhanced metastatic potential. Calcipotriol treatment of enhanced metastatic, but not parental, cells reduces proliferation and survival via vitamin D receptor (VDR) signaling, increases the expression of RASSF2, a negative regulator of the Hippo signaling pathway, and reduces the levels of the Hippo pathway effectors YAP and TAZ. RASSF2 is required for the effects of calcipotriol and for the reduction of levels and nuclear localization of YAP/TAZ. Migration of the enhanced metastatic cells and YAP/TAZ levels are reduced after calcipotriol treatment and YAP overexpression reduces calcipotriol sensitivity. Furthermore, metastatic cells that overexpress VDR also showed lower tumor burden in vivo. Implications: This newly identified link between VDR signaling and the Hippo pathway could inform treatment strategies for metastatic neuroblastoma.

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Supplementary Figure from Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma