The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

MYC oncoproteins deliver a potent oncogenic stimulus in several human cancers, making them major targets for drug development, but efforts to deliver clinically practical therapeutics have not yet been realized. In childhood cancer, aberrant expression of MYC and MYCN genes delineates a group of aggressive tumours responsible for a major proportion of pediatric cancer deaths. We designed a chemical-genetic screen that identifies compounds capable of enhancing proteasomal elimination of MYCN oncoprotein. We isolated several classes of compound that selectively kill MYCN expressing cells and we focus on inhibitors of PI3K/mTOR pathway in this study. We show that PI3K/mTOR inhibitors selectively killed MYCN-expressing neuroblastoma tumor cells, and induced significant apoptosis of transgenic MYCN-driven neuroblastoma tumors concomitant with elimination of MYCN protein in vivo. Mechanistically, the ability of these compounds to degrade MYCN requires complete blockade of mTOR but not PI3 kinase activity and we highlight NVP-BEZ235 as a PI3K/mTOR inhibitor with an ideal activity profile. These data establish that MYCN expression is a marker indicative of likely clinical sensitivity to mTOR inhibition, and provide a rationale for the selection of clinical candidate MYCN-destabilizers likely to be useful for the treatment of MYCN-driven cancers.

Purpose-To evaluate noninvasive and clinically-translatable magnetic resonance imaging (MRI) biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of aggressive, MYCN-amplified neuroblastoma.Materials and methods-All experiments were performed in accordance with the local ethical review panel, the UK Home Office Animals Scientific Procedures Act 1986 and with the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research. Multiparametric MRI was performed on abdominal tumors found in the TH-MYCN model. T 2 -weighted MRI, quantitation of native relaxation times T 1 and T 2 , the relaxation rate R 2 *, and dynamic contrast-enhanced (DCE) MRI were used to monitor tumor response to cyclophosphamide (25mg/kg), the vascular disrupting agent ZD6126 (200mg/kg), or the antiangiogenic agent cediranib (6mg/kg, daily). Any significant changes in the measured parameters, and in the magnitude of the changes upon treatment between treated and control cohorts, were identified using Student's 2-tailed paired and unpaired t-test respectively, with a 5% level of significance. Conclusions-The T 1 relaxation time is a robust noninvasive imaging biomarker of response to therapy in tumors in TH-MYCN mice, which emulate high-risk neuroblastoma in children. T 1 measurements can be readily implemented on clinical MR systems, and should be investigated in translational clinical trials of new targeted therapies for pediatric neuroblastoma. Results-Treatment

Concomitant inhibition of anaplastic lymphoma kinase (ALK) and bromodomain-4 (BRD4) is a potential therapeutic strategy for targeting two key oncogenic drivers that co-segregate in a significant fraction of high-risk neuroblastoma patients, mutation of ALK and amplification of MYCN. Starting from known dual polo-like kinase (PLK)-1–BRD4 inhibitor BI-2536, we employed structure-based design to redesign this series toward compounds with a dual ALK–BRD4 profile. These efforts led to compound (R)-2-((2-ethoxy-4-(1-methylpiperidin-4-yl)phenyl)amino)-7-ethyl-5-methyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5H)-one (16k) demonstrating improved ALK activity and significantly reduced PLK-1 activity, while maintaining BRD4 activity and overall kinome selectivity. We demonstrate the compounds’ on-target engagement with ALK and BRD4 in cells as well as favorable broad kinase and bromodomain selectivity.