Valencia-Sama, I.;Kee, L.;Weiss, A.;Hayes, M.;Ohh, M.;Irwin, M.

Metastatic neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, has a cure rate of <50%. DNA-sequencing studies have demonstrated rare recurrent driver mutations at diagnosis, with the most common alterations detected in ALK-RAS-MAPK pathway. Activating ALK and RAS-MAPK mutations are associated with inferior outcome and are increased at relapse, and thus, represent therapeutic vulnerabilities in NB. Previously, we identified combinations of RAS/MAPK inhibitors, including SHP2 and MEK, with efficacy in resistant MAPK-altered tumor cells, including those with the most common NB-associated RAS mutation NRAS-Q61K. However, toxicities of SHP2 inhibitors and promising results using compounds that directly target RAS suggest there may be superior strategies to target RAS/MAPK pathway in NB. Here, we have assessed the efficacy of RAS/MAPK inhibitors, including tovorafenib (pan-RAF), RMC-6236/daraxonrasib (pan-active-RAS) and avutometinib (RAF/MEK) in NB in vitro and in vivo using NB models harboring differing genomic status of RAS/MAPK pathway effectors. We demonstrate selective efficacy of RMC-6236 and avutometinib via RAS-MAPK pathway inhibition in NB cells and xenografts harboring RAS, NF1 or ALK alterations. Importantly, we demonstrate that presence of the NRAS-Q61K mutation confers drug sensitivity. Using newly generated and previously established NB cell models of acquired resistance to RMC-6236 or the ALK inhibitor lorlatinib, we identified targeted combinations, including RMC-6236 plus avutometinib, that demonstrate re-sensitization in resistant NB cell and xenograft models. Finally, transcriptomic studies of RMC-6236-resistant cells detected upregulation of RAS/MAPK signatures, as well as TNFα/NFκB and IL-6/JAK/STAT3 pathway enrichment, thus informing future combinations to enhance sensitivity to RAS inhibitors. STATEMENT OF SIGNIFICANCE Our work demonstrates that newly available RAS pathway inhibitors RMC-6236/daraxonrasib and avutometinib have efficacy in neuroblastoma tumors, which have frequent alterations in the RAS/MAPK pathway. These drugs with early efficacy results in adult RAS-driven tumors provide an important option for patients with relapsed neuroblastoma alone or in combination.