Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma.

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.

NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS-MAPK Pathway in Neuroblastoma.

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 NRASQ61K 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 therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.