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[46]
ALK inhibition with up-front chemotherapy in ALK-positive neuroblastoma patients. Krytska et al.
demonstrated that neuroblastoma ALK cell lines with de novo resistance to Crizotinib, achieved complete
responses to Crizotinib combined with chemotherapy in xenograft models. This synergy was dependent
upon a functional p53 pathway, further making the case for targeted therapy upfront in treatment
[47]
schedules, as p53 pathway in-activation is a characteristic of chemotherapy-resistant disease . The
phase II pediatric study, PEDS-PLAN, is currently evaluating feasibility of molecularly guided therapy in
combination with induction chemotherapy (NCT02559778). Additionally, Crizotinib is being tested against
Iobenguane I-131 with standard therapy for children newly diagnosed with high-risk neuroblastoma or
ganglioneuroblastoma (NCT03126916).

In ALK-rearranged NSCLC, there is a strong precedent for the transition of patients between different ALK
inhibitors, as a common mechanism of resistance in this patient group is the acquisition of a secondary
[48]
treatment-induced ALK mutation . It has even been reported that re-challenge with a previously used
[49]
ALK inhibitor can be effective, if compound ALK mutations occur with different inhibitor sensitivities .
In neuroblastoma, the picture of acquired resistance to ALK inhibitors may be very different. Upregulation
of signaling through the alternative tyrosine kinase AXL, associated with an increase in endothelial-to-
mesenchymal transition, has been proposed in response to induced resistance to the non-clinical ALK
[50]
inhibitor TAE684, in vitro . A more recent study of induced Lorlatinib resistance in vitro pin-pointed an
[51]
acquired mutation of NF1 with RAS/MAPK activation .
In fact, the dominance of the RAS/MAPK pathway in refractory and relapsing neuroblastoma is
becoming clear. Relapsed high-risk neuroblastoma has a higher mutational burden than neuroblastomas
at diagnosis, and the largest study of this type has shown that over half of the mutations identified at
[52]
relapse are targetable by compounds already in clinical development . This study also concurred with
evidence of the preponderance of RAS/MAPK pathway mutations at relapse published previously [53-55] .
[54]
For example, Eleveld et al. carried out a whole genome sequencing paired study of 23 diagnostic and
relapsing neuroblastomas, finding that 18 out of 23 relapse samples had acquired mutations predicted to
hyperactivate the RAS/MAPK pathway. Included in this relapse group were 10 mutations of ALK, strongly
suggesting that the incidence of ALK mutations at relapse is higher than at diagnosis. Re-sequencing of
tumors in relapsing patients is therefore essential, due to the availability of ALK inhibitors, amongst others.
However, the application of MEK inhibitors to treat ALK-activated neuroblastomas is not straightforward.
As demonstrated in a recent preclinical study, MEK/ERK inhibition in this context results in the increased
[56]
activation of AKT-ERK5 and therefore does not slow neuroblastoma growth . The complexity of the
signaling, and escape-signaling pathways involved in neuroblastomas treated with targeted inhibitors
underlines the need for a personalized medicine approach to treat these patients.

In anticipation of resistance to ALK inhibition and in order to improve the best response for patients with
ALK mutant neuroblastoma, combination clinical studies are already underway. The NEPENTHE (Next
Generation Personalized Neuroblastoma Therapy, NCT02780128) study is designed to match genomic
aberrations found at neuroblastoma relapse with the optimal combination of small molecule targeted
treatment. ALK-positive patients enrolled onto this study will receive a combination of Ceritinib with the
CDK 4/6 inhibitor, Ribociclib. This combination demonstrated strong evidence of synergy in preclinical
work, using both in vitro experiments to study the effect of the combination on the relevant cyclin D/
CDK4/CDK6/RB and ALK signaling pathways, and in vivo trials with conventional and patient-derived
[57]
xenograft models . The precise mechanism of interaction between these two compounds is not fully
characterized, except that when dosed together in ALK mutant cell lines, there was enhanced depletion of
both phosphorylated ALK and phosphorylated Rb, compared to either agent alone.