The Next-Generation Alk Inhibitor Pf-06463922 Overcomes Primary Resistance To Crizotinib In Neuroblastoma

Neuroblastomas (NBs) harboring activating point mutations in the kinase domain of anaplastic lymphoma kinase (ALK) are differentially sensitive to ALK inhibition by crizotinib (Bresler et al. 2011). The F1174L and F1245C amino acid substitutions, which comprise 38.5% (mycancergenome.org) of ALK aberrations in NB, also confer intrinsic resistance to crizotinib and present a significant obstacle in the clinic. Therefore, our goal has been to identify a next-generation ALK inhibitor with improved selectivity and potency to target these resistant mutants effectively. We demonstrate here that PF-06463922 (Pfizer), an ATP-competitive, macrocyclic ALK/ROS1 inhibitor, overcomes crizotinib resistance of these ALK variants in NB, and exerts unprecedented activity as a single agent against the F1174L and F1245C ALK-mutated tumors. At a tenfold lower dose (10 mg/kg) than typically used for crizotinib (100 mg/kg), PF-06463922 induces complete tumor regression in patient-derived (COG-N-426x, F1245C ALK-mutated) and cell line-derived (SY5Y, F1174L ALK-mutated) xenograft mouse models. Whereas crizotinib-treated mice in both models experience rapid tumor growth on therapy, PF-06463922-treated mice showed sustained complete responses for several weeks even after treatment was discontinued. Cell-based and biochemical analyses indicate that the superior activity of PF-06463922 is a function of both higher potency and a broader spectrum of effectiveness across mutants. ATP levels were measured in ten NB cell lines upon treatment with PF-06463922 and crizotinib; at 120 hours post-treatment, PF-06463922 is 26-38 times more effective at inducing growth inhibition in cells with primary resistance than crizotinib. These in vitro and in vivo results are corroborated by our biochemical experiments. In comparative kinase inhibition studies of several next-generation ALK inhibitors, PF-06463922 was most effective against frequently observed ALK variants accounting for approximately 75% of neuroblastoma samples, including those with F1174L mutations. In vitro, PF-06463922 showed at least 3-4 fold higher potency than crizotinib in peptide phosphorylation assays, and its superior inhibition was consistently observed in cellular analysis of transforming ability. Further investigation is necessary to elucidate PF-064639229s mechanism of action and reveal how it induces apoptotic signaling networks without abrogating phospho-ALK. Taken together, these results support the argument that a modified ALK inhibitor may improve outcomes for patients with primary resistance to crizotinib and provide the preclinical basis from which to design a clinical trial for PF-06463922 for the treatment of ALK-mutated NBs. Citation Format: Nicole Infarinato, Jin Park, Renata Sano, Kateryna Krytska, Hannah Ryles, Helen Zou, Nathan Lee, Tod Smeal, Mark A. Lemmon, Yael P. Mosse. The next-generation ALK inhibitor PF-06463922 overcomes primary resistance to crizotinib in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2015-1619