Abstract A31: Activation of the RAS-MAPK pathway in primary neuroblastoma tumors is associated with poor prognosis
Authors: Eleveld T.F. , Schild L , Ebus M.E. , Sluis P.G. van , Westerhout E.M. , Caron H.N. , Koster J.J.B. , Versteeg R. , Molenaar J.J. 
Published: May 2016

We recently identified that mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma, which suggests that activation of this pathway may play an important role in the aggressive phenotype of these tumors. Furthermore, it was shown that the presence of these mutations may serve as a biomarker for treatment with MEK inhibitors both in vitro and in vivo. These results suggest that MEK inhibitors may be a viable treatment option for aggressive neuroblastoma tumors and warrant further investigation into the role of the RAS-MAPK pathway in neuroblastoma.

To identify if, and to what extent, the RAS-MAPK pathway is also activated in primary neuroblastoma tumors we have developed a RAS-MAPK mRNA signature. Genes were included that were I) regulated by mutant ALK, which is a known activator of the RAS-MAPK pathway in neuroblastoma, and II) were conversely regulated by treatment with the MEK inhibitor U0126. We ended up with a core signature consisting of 8 genes, which was tested in a panel of 17 neuroblastoma cell lines. The expression of our gene signature in this panel correlates with mutations in the RAS-MAPK pathway, with the expression of phospho-MEK and phospho-ERK on Western Blot and with sensitivity to the MEK inhibitor Trametinib.

Clustering analyses in our set of 122 primary neuroblastoma tumors shows that high expression of this signature is associated with significantly poorer survival and similar analyses in other publicly available datasets corroborate these results, even if applied to only stage 4 tumors . In our data set all tumors containing mutations in known RAS-MAPK pathway genes, such as ALK and NF1, show high signature scores, which makes it likely that this signature can indeed detect the activation state of the pathway through mRNA profiling.

However, there are also tumors with high signature scores that do not show mutations that are known to be associated with RAS-MAPK signaling (as detectable by Whole Genome Sequencing). Therefore we looked in these tumors if there were other mutations that could explain activation of this pathway. We detected mutations in known RAS-MAPK genes, such as SOS1, but also recurrent aberrations in genes not previously known to be related to RAS-MAPK signaling, such as PHOX2B, DMD and ERF. We are currently characterizing the effect of these mutations on the RAS-MAPK signaling pathway in neuroblastoma cell lines.

Our findings show that activation of the RAS-MAPK pathway, as determined by our mRNA gene signature, is associated with poor prognosis in neuroblastoma. In some tumors this activation is caused by known activating mutations in this pathway, but in most cases the causative events remain unknown. We present several other candidate genes, which when mutated may also lead to pathway activation. MEK inhibitors were already shown to be effective against neuroblastoma xenografts with RAS or NF1 mutations, and our results indicate that a broader selection of neuroblastoma tumors might respond to these compounds.

Citation Format: T.F. Eleveld, L Schild, M.E. Ebus, P.G. van Sluis, E.M. Westerhout, H.N. Caron, J.J.B. Koster, R. Versteeg, J.J. Molenaar. Activation of the RAS-MAPK pathway in primary neuroblastoma tumors is associated with poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A31.