87.06 Dual Target Inhibition of RET and the RAS-MAPK Pathway with RXDX-105 as a Novel Therapeutic in Neuroblastoma

S. Flynn¹, J. Lesperance², N. Phanhthilath², M. Paul², P. Zage^{2  1}University Of California – San Diego,Surgery,San Diego, CA, USA ²University Of California – San Diego,Pediatrics,San Diego, CA, USA

Introduction:

Novel therapies are needed for high risk neuroblastoma, where outcomes remain poor despite aggressive multimodal therapy. The RET tyrosine kinase is highly expressed in neural crest and neuroblastoma cells and is felt to play a role in their proliferation and survival. BRAF mutations are uncommon, but have been described in neuroblastoma tumors, and Eleveld et al. demonstrated high rates of RAS-MAPK mutations in relapsed neuroblastoma. RXDX-105 is a small molecule multikinase inhibitor with potent activity against RET and BRAF, currently under Phase I/Ib clinical trials in adult solid tumors. Given the suggested role of RET and the RAS-MAPK pathway in neuroblastoma survival, proliferation, and relapse, we investigated dual inhibition of these pathways with RXDX-105 as a potential new therapeutic for neuroblastoma.

Methods:
The effect of RXDX-105 on cell proliferation and viability was evaluated using Incucyte Zoom^TM continuous live cell imaging and alamarBlue® assays. Caspase 3/7 cleavage assays were used to asses for apoptosis. Pathway inhibition effects on cell cycle were measured using flow cytometry with propidium iodide staining. Inhibition of RET and the RAS-MAPK pathways were measured with Western blot analysis of treated and untreated cells. The efficacy of RXDX-105 against neuroblastoma tumors in vivo was evaluated with an orthotropic xenograft model in immunocompromised mice treated with oral RXDX-105. Tumors were assessed with in vivo luminescence and final tumor weight.

Results:
IC₅₀ values for the tested cell lines ranged between 5mcM and 14mcM following 72 hours of RXDX-105 exposure. Drug treated cells showed an increase in caspase 3/7 cleavage as well as G1 cell cycle arrest. RXDX-105 treatment potently inhibited RET phosphorylation as well as phosphorylation of downstream proteins of BRAF, including MEK and ERK. Daily oral treatment with RXDX-105 in vivo resulted in reduced tumor growth.

Conclusion:
Inhibition of RET and BRAF/RAS-MAPK pathway inhibits neuroblastoma cell growth and viability through combination of induction of apoptosis and cell cycle arrest. Inhibition of these pathways with RXDX-105 shows promise as a novel therapeutic in preclinical xenograft models.