Combination therapies targeting alk-aberrant neuroblastoma in preclinical models

Background ALK activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1-2% of cases. Lorlatinib, a third generation ALK inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. Aims To study the preclinical activity of ALK inhibitors alone and in combination with chemotherapy or idasanutlin. Methods We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX). Results Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK -amplified PDX model with the highest ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. Conclusion Our study suggests that in neuroblastoma, high ALK expression could be associated with response to lorlatinib and either chemotherapy or idasanutlin. The synergy between MDM2 inhibition and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma. STATEMENT OF TRANSLATIONAL RELEVANCE Neuroblastoma is a pediatric tumor of the developing sympathetic nervous system. Around 50% of high-risk neuroblastoma patients are curable. Mutations or amplification of Anaplastic Lymphoma Kinase (ALK) have emerged as a marker with which to further risk-stratify patients. The ALK inhibitor lorlatinib will soon be used alongside chemotherapy in upfront treatment of high-risk patients with ALK-aberrant disease. In this preclinical study, we used a panel of ALK aberrant neuroblastoma models to evaluate ALK inhibitors focusing on lorlatinib in combination with conventional chemotherapy and the small molecule MDM2 inhibitor idasanutlin. In both approaches we found synergy in models with high basal ALK expression without MAPK pathway alterations. We conclude that in neuroblastoma the level of ALK expression could be an additional biomarker predictive of clinical response to ALK inhibitors. ### Competing Interest Statement The authors have declared no competing interest.