The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

Kinases such as MEK are attractive targets for novel therapy in cancer, including acute myeloid leukaemia (AML). Acquired and inherent resistance to kinase inhibitors, however, is becoming an increasingly important challenge for the clinical success of such therapeutics, and often arises from mutations in the drug-binding domain of the target kinase. To identify possible causes of resistance to MEK inhibition, we generated a model of resistance by long-term treatment of AML cells with AZD6244 (selumetinib). Remarkably, resistance to MEK inhibition was due to acquired PTEN haploinsufficiency, rather than mutation of MEK. Resistance via this mechanism was confirmed using CRISPR/Cas9 technology targeting exon 5 of PTEN. While PTEN loss has been previously implicated in resistance to a number of other therapeutic agents, this is the first time that it has been shown directly and in AML.

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.

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

Celyvir (autologous mesenchymal cells -MSCs- that carry an oncolytic adenovirus) is a new therapeutic strategy for metastatic tumors developed by our research group over the last decade. There are limitations for studying the immune effects of human oncolytic adenoviruses in murine models since these viruses do not replicate naturally in these animals. The use of xenografts in immunodeficient mice prevent assessing important clinical aspects of this therapy such as the antiadenoviral immune response or the possible intratumoral immune changes, both of tumor infiltrating leukocytes and of the microenvironment. In our strategy, the presence of MSCs in the medicinal product adds an extra level of complexity. We present here a murine model that overcomes many of these limitations. We found that carrier cells outcompeted intravenous administration of naked particles in delivering the oncolytic virus into the tumor masses. The protection that MSCs could provide to the oncolytic adenovirus did not preclude the development of an antiadenoviral immune response. However, the presence of circulating antiadenoviral antibodies did not prevent changes detected at the tumor masses: increased infiltration and changes in the quality of immune cells per unit of tumor volume, and a less protumoral and more inflammatory profile of the tumor microenvironment. We believe that the model described here will enable the study of crucial events related to the immune responses affecting both the medicinal product and the tumor.