Background In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research. Methods To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX . We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF. Findings In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft. Interpretation ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic. Funding This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies.

Targeted inhibition of anaplastic lymphoma kinase (ALK) is a successful approach for the treatment of many ALK-aberrant malignancies; however, the presence of resistant mutations necessitates both the development of more potent compounds and pharmacodynamic methods with which to determine their efficacy. We describe immunoassays designed to quantitate phosphorylation of ALK, and their use in preclinical models of neuroblastoma, a pediatric malignancy in which gain-of-function ALK mutations predict a poor overall outcome to conventional treatment. Validation of the immunoassays is presented using a panel of neuroblastoma cell lines and evidence of on-target ALK inhibition provided by treatment of a genetically engineered murine model of neuroblastoma with two clinical ALK inhibitors, crizotinib and ceritinib, highlighting the superior efficacy of ceritinib.

10036 Background: Preclinical data in neuroblastoma (NBL) models provided evidence that the addition of an mTOR inhibitor with crizotinib may overcome the relative resistance to ALK inhibitors (Berry et al., 2012, Cancer Cell). The primary objective of this phase 1b trial was to establish the recommended phase 2 dose (RP2D) of crizotinib in combination with the mTOR inhibitor temsirolimus in pediatric NBL patients. Methods: Patients aged 1-21 years with relapsed/refractory ALK/MET aberrated NBL were eligible to enroll onto stratum 2 of the CRISP trial (EudraCT: 2015-005437-53, ITCC-053), sponsored by Erasmus MC. This was an open-label, non-randomized, two-stage phase 1b dose finding study. Crizotinib was dose escalated using the EWOC method in four dose levels (DL) ranging from 100 mg/m2 to 280 mg/m2 twice daily (BID) in 28 day cycles. Temsirolimus was administered intravenously in a fixed dose of 60 mg/m2/week. Both starting doses were reduced compared to the single agent RP2D because of an expected CYP3A4 interaction. Primary endpoint was dose limiting toxicity (DLT) during cycle 1. Radiological disease assessments and blood sampling for pharmacokinetics were scheduled per protocol. Results: Nine NBL patients were enrolled onto stratum 2 between 06-2018 and 12-2022. Eight patients with a median age of 7 years (range: 1-12) received trial treatment. The temsirolimus dose was reduced to 40 mg/m2 after the first 3 patients based on unexpected toxicity. DLTs occurred in 2/3 patients treated with crizotinib at DL2 (200 mg/m2 BID); grade 3 elevated ALT (alanine aminotransferase) and grade 3 gastritis (temsirolimus dose; see table). No DLT occurred in 5 patients treated with crizotinib DL1 (150 mg/m2 BID). Dose re-escalation of crizotinib to DL2 was considered not feasible. The RP2D was established at 150 mg/m2 BID crizotinib with 40 mg/m2 temsirolimus. Frequent grade 3 treatment-related adverse events were anorexia, gastritis, oral mucositis (n = 2) and anemia (n = 3). Treatment, toxicity and response are summarized in table. Conclusions: We established a RP2D of 150 mg/m2 BID crizotinib in combination with 40 mg/m2 temsirolimus. The expansion cohort is currently open for enrollment. Clinical trial information: EUCTR2015-005437-53 . [Table: see text]