The clinical outcome of children with high-risk neuroblastoma, a cancer of the sympathetic nervous system, remains low. All neuroblastoma survivors have high-risk of developing long-term disabilities and life-threatening conditions caused by current treatments. Better and safer therapies are urgently needed.¹The proto-oncogene MYCN plays a central role in the biology of high-risk neuroblastoma and represents a major therapeutic target. Aurora A Kinase is an enzyme responsible for the stabilisation of the MYCN protein. Inhibition of Aurora A kinase activity with the small molecule inhibitor MLN8237 (Alisertib, Millennium Pharmaceuticals Inc.) results in ablation of mycn and significant anti-tumor activity in a genetically-engineered mouse (GEM) model of neuroblastoma. MLN8237 is currently being evaluated in early-phase pediatric clinical trials.² The use of pharmacodynamic (PD) biomarkers has accelerated the clinical development of novel therapeutics against adult cancer. Conventional PD biomarkers necessitate access to post therapy biopsies, which is often not possible in the pediatric population. Therefore non-invasive alternatives, such as imaging biomarkers must be pursued. We have shown that GEM models of neuroblastoma that faithfully recapitulate the major genetic, patho-physiological and radiologic features of the childhood disease, represent an information-rich platform with which to evaluate novel therapeutic strategies and associated noninvasive imaging biomarkers. ^3,4We have previously demonstrated that a decrease in tumor native spin lattice relaxation time T₁ is a biomarker of response to both cyclophosphamide, an ubiquitous component of frontline treatment for children with neuroblastoma, and vascular-targeted therapies in the Th-MYCN GEM model of neuroblastoma. The aim of this study was to evaluate if T₁ provides a biomarker of response to targeted therapy with MLN8237 in the Th-MYCN model and evaluate the hypothesis that the decrease in T₁ is due to a drug-induced increase in ferric iron Fe(III). All experiments were performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 and the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research.⁵ Mice with abdominal tumors were identified by palpation. On day 1, mice were treated with 30mg/kg p.o. with MLN8237 (n=8) or vehicle (n=7). MRI was performed on day 0 and day 2 (24h after treatment started). MRI studies were performed on a 7T Bruker MicroImaging system using a 3cm birdcage volume coil. T₂-weighted axial images were used for determining tumor volumes and, following optimization of the local field homogeneity (Fastmap), to plan the T₁ measurement (inversion recovery True-FISP sequence, FOV 3x3cm², 128x128 phase encoding steps, TI= 28-1930ms, 50 inversion times, TE=1.2ms, TR=2.5ms, scan TR=10s, 8 segments, NEX=8), as previously described.⁴ Histology. Formalin-fixed paraffin-embedded sections from tumors treated with either MLN8237 or vehicle control, as well as historical sections from tumors treated with cyclophosphamide ( 25mg/kg, 48h post treatment), were stained with hematoxylin and eosin (H&E) for the assessment of cell death and Perls’ Prussian Blue staining for Fe(III). Treatment with MLN8237 led to significant reduction in tumor burden in the Th-MYCN model. MLN8237 anti-tumor activity was associated with a significant global decrease in native T₁, 24 hours after treatment started (Fig.1 & 2). Despite significant tumor progression in the vehicle cohort, T₁ remained constant. H&E staining showed increased numbers of cells with condensed nuclei indicative of cell death (Fig.3, black arrow) in both MLN8237- and CPM-treated tumors compared to control. Perls’ staining showsed increased concentration of Fe(III) within the dying cells (Fig.3, white arrow). This study demonstrates that T₁ is a biomarker of tumor response to MLN8237 in the Th-MYCN model of neuroblastoma, and reinforces T₁ as a generic biomarker of successful response to therapy in this model. T₁ quantification is rapid and completely noninvasive, and based on our previous work, has already been incorporated routinely into early phase MRI-embedded clinical trials of novel therapies for children with neuroblastoma at the Royal Marsden Hospital (UK). Furthermore our study strongly suggests that T₁ is a biomarker of cell death in this model, due to its sensitivity to an increased concentration of superparamagnetic Fe(III) within the dying cells.Further investigation is needed to understand the origin of the increase in Fe(III) upon cell death. Iron is essential to neuroblastoma cell proliferation and iron chelators have shown anti-tumor effect in children with neuroblastoma.⁶ An increase in ferritin, the major iron storage protein (Fe(III)), has been reported in childhood neuroblastoma following chemotherapy.⁷T₁ is a biomarker of response to Aurora A kinase inhibitor MLN8237, and a generic biomarker of cell death in the Th-MYCN model of neuroblastoma.