In this issue Mossé and coworkers report the results of preclinical testing of a novel ALK/ROS1 inhibitor PF06463922 in neuroblastoma. relapse of fatal therapy-resistant lesions. Since the original identification of activating somatic mutations in neuroblastoma in 2008 multiple large-scale sequencing studies have established a consensus mutation Amiloride HCl rate of approximately 8% with amplification of ALK comprising another 4%. Studies on the prognostic impact of ALK mutations have been conflicting while others have found that ALK overexpression supersedes mutations in predicting outcome. Three types of kinase domain mutations are dominant – F1174L R1275Q and F1245C – all of which confer increased proliferation growth factor independence and activation of canonical downstream signaling pathways. These changes induce tumor development in nude mice thus firmly establishing the oncogenic role of mutant ALK in neuroblastoma. The ALK F1174L mutation has attracted much attention primarily because of its Amiloride HCl cosegregation with MYCN amplification in human tumors and an enhanced tumorigenicity in transgenic animals (1 2 As hardly any other mutated kinases had been identified in neuroblastoma the discovery of ALK mutations in 2008 generated much hope for targeted therapy of this tumor and enthusiasm was high for the immediate translation of this finding. This led to the rapid institution of a Children’s Oncology Group (COG) Phase 1 trial with the only clinically available inhibitor with activity against ALK crizotinib. This drug had shown remarkable activity in patients with non small cell lung cancer (NSCLC) characterized by expression of oncogenic ALK fusion proteins. However in preclinical studies in neuroblastoma it became clear that while crizotinib inhibited growth and induced apoptosis in cells expressing ALK R1275Q it failed to inhibit the growth of ALK F1174L-positive cells (3). Further F1174L was one of the resistance mutations that arose in adult cancer patients treated with crizotinib as a single-agent (4). This deficiency was illustrated in the COG trial of crizotinib where neuroblastoma patients with point mutations in mutations. Four models were tested two PDX models expressing F1174L and F1245C respectively and two established neuroblastoma cell line xenograft models expressing F1174L and R1275Q all of which were treated for a minimum of 6 weeks. PF06463922 induced a shrinkage of tumor volumes below palpable detection in all four models starting from 2–3 weeks after the onset of treatment. Downregulation of ALK phosphorylation was shown only in the R1275Q xenograft model. In three models the tumors remained undetectable during the full 6 to 9 weeks of treatment. In the fourth model (R1275Q) a small tumor emerged 7 to 8 weeks after the start of treatment. While this is a major improvement over responses obtained with crizotinib the data also predict the limitations of the drug. Discontinuation of PF06463922 resulted in regrowth of the tumors within 4 to 7 weeks in all 4 models suggesting that in the clinical setting a population of tumor cells will likely persist during treatment and ultimately give rise to relapse (8). The nature of the recurrent tumors was not investigated by Mossé and coworkers. The tumors were followed by palpation only which precludes an accurate estimate of the amount of viable tumor persisting during treatment. Additionally in the in vitro studies while the IC50 values were significantly better than those for crizotinib PF06463922 appeared to inhibit the growth of only a proportion of the cells with as many as 25–50% remaining at maximum drug concentrations. Whether these remaining cells undergo Amiloride HCl growth arrest or senescence is not addressed by the data presented. It is possible that the drug leaves a residual subpopulation of inherently resistant cells that enter a slow cycling state only to rapidly proliferate after the drug stimulus is removed. This phenomenon of tumor cell plasticity Rabbit Polyclonal to ARF6. in the presence of certain therapeutic agents (9) may well account for recurrences seen in the in vivo models described in this study. The fact that PF06463922 on the Amiloride HCl other hand causes complete growth inhibition of NSCLC cells expressing EML4-ALK and NIH3T3 cells transfected with the three neuroblastoma-associated ALK mutations further supports the premise that neuroblastoma tumors may contain a subpopulation of cells that are inherently resistant to PF06463922. The.