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10024 Background: KMT2a rearrangements are a hallmark of infant (less than 1 year of age) leukemia and are associated with poor prognosis. Menin is a ubiquitous protein that binds to the N-terminal of the KMT2a-fusion protein to mediate the oncogenic activity of KMT2a-rearranged leukemia cells. In this study, we evaluated the effect of multiple menin inhibitors and effective drug combinations for the treatment of KMT2A-rearranged infant leukemia. Methods: FISH analysis and immunoblotting confirmed the presence of KMT2a-fusion in the primary patient samples and cell lines studied. Lymphocytes from healthy donors were used as controls. Cells were treated with various concentration of multiple menin inhibitors for 72 hours and growth inhibition was measured using alamar blue assay. Infant leukemia cells were treated with a panel of FDA-approved agents (n = 221) to identify potential synergy, additive- and antagonistic-effects with specific menin inhibitors. Therapeutic drug interaction properties were established by calculating combination indices (CI) using the Chou-Talalay method. Mode of cell death and cellular target modulations were determined by western blot analysis. The effect of targeting menin in the context of the leukemogenic bone marrow microenvironment was determined through stromal cell co-cultures. Results: A comprehensive analysis of menin inhibitors on infant leukemia cell lines and primary patient cells exhibited significant and quantitatively diverse responses in cells with distinct molecular properties. Within the panel of menin inhibitors, infant B-ALL cells were found to be more sensitive to MI-463, MI-503 and MI-136 with a mean IC50 of 5.9µM, 6.1µM and 10.2µM, respectively. On the other hand, MI-3 exhibited an IC50 of 35.6µM. The cytotoxic effect of menin inhibitors on normal lymphocytes was minimal, suggesting a favourable therapeutic window (p < 0.0001). High-throughput screening with a library of > 200 FDA-approved drugs revealed significant sensitivity of distinct infant leukemia cells to proteasome, HDAC and CDK9 inhibitors. Among these, substantial drug synergy was observed between menin and proteasome inhibitors. For example, carfilzomib synergised with menin inhibitors over a broad range of concentrations with a CI value of 0.7. Conclusions: In this study, we present and discuss the initial proof-of-concept preclinical data for the effective anti-leukemic activity of menin inhibition against KMT2A-rearranged infant leukemia cells. Furthermore, the comprehensive drug screen and drug combination studies identified a spectrum of mechanistically-validated synergies, providing usable data for the formulation of multi-agent clinical studies for this currently unmet need in pediatric oncology.

10035 Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite the development of risk stratification tools to improve prognostication, prediction of patient survival outcomes in NB remains poor. In this study we used an unbiased machine-learning algorithm to develop and validate a transcriptomic signature capable of predicting 5-year overall (OS) and event-free survival (EFS) in these patients. Methods: The TARGET-Neuroblastoma dataset (n = 243) was used as the training set. Two independent NB cohorts, E-MTAB-179 (n = 478) and GSE85047 (n = 266) were used as validation sets. Elastic net regression was employed to identify transcripts associated with EFS. Association of the developed signature with EFS and OS was evaluated using univariate Cox proportional hazards (CoxPH), Kaplan-Meier, and 5-year receiver-operator characteristic curves in validation cohorts. Further, the independent prognostic value of the signature was assessed using multivariate CoxPH models with relevant clinicopathologic variables including age, INSS stage, and N-Myc amplification status in both validation sets. Finally, a nomogram was developed to integrate the signature with prognostic clinicopathologic variables to evaluate their combined efficacy for prediction of 5-year EFS and OS. Results: We identified a 21-gene signature that demonstrates significant association with EFS and OS in both E-MTAB-178 and GSE49710 validation cohorts. Moreover, the signature is independent of clinicopathological variables and can be effectively incorporated into a risk model, improving the prognostic performance. Several genes within the signature have been previously implicated in NB, including ECEL1, HOXC9 and ARAF1. Conclusions: To the best of our knowledge, we are the first to use an unbiased machine learning approach to generate a transcriptomic gene signature for neuroblastoma prognosis externally validated in multiple cohorts across platforms. This 21-gene transcriptomic signature significantly associated with EFS and OS in this disease. Combining this signature with current prognostic clinicopathologic variables will improve risk stratification of affected patients and may inform effective clinical decision-making.[Table: see text]

10033 Background: Although survival rates have improved in the recent past, relapse and refractory disease remain a significant cause of death in children with leukemia. This calls for an urgent need for the development of novel therapies that could effectively treat leukemias in children. The E26 transformation specific (ETS) family of transcription factors regulate various normal cellular functions but are abnormally expressed in various cancers, including leukemia. TK216 is an ETS inhibitor, that has shown pre-clinical activity and clinical efficacy in solid tumors. In this study, we explore the feasibility of using TK216 as a therapeutic agent for the treatment of high risk refractory pediatric leukemia. Methods: A panel of pediatric leukemia derived cell lines and primary blast cells representing a spectrum of molecular abnormalities seen in pediatric leukemia were treated in vitro with TK216 to determine cytotoxicity. Normal lymphocytes were used as controls and cell viability was determined 72 hours post-treatment by Alamar blue assay. The induction of tumor cell apoptosis and target modulation were detected by Western blotting. Alterations in the cell cycle were assessed by FACS analysis with PI staining. Drug combination studies were carried out with established anti-leukemic agents to identify synergy for greater therapeutic efficiency. Results: TK216 decreased cell viability in leukemia cells compared to normal lymphocyte controls in a dose-dependent manner with variations in sensitivity noted with inherent molecular abnormalities. The IC50 values observed ranged from 0.22 µM for the most sensitive cell line, MV4-11 to 0.95 µM for least sensitive cell line, SUP-B15. Apoptosis induction upon TK216 treatment was confirmed by PARP cleavage and caspase 3 activation. Cell cycle analysis demonstrated increased sub-G1 population of cells after TK216 treatment. A strong correlation between sub-G1 population and sensitivity of the cell line towards TK216 (47% in MV4-11 vs 3.72% in SUP-B15) was observed. Screening of a panel of 200 FDA approved anti-cancer agents in drug combination studies identified potential agents for drug synergy. Significant drug synergy was noted with TK216 in combination with the epigenetic modifier 5-azacytidine and the Bcl-2 inhibitor, Venetoclax. [Combination Index for Venetoclax and TK216, mean = 0.65 for MV4-11 and 0.33 for SUP-B15]. Conclusions: Data from our study demonstrate that the ETS inhibitor TK216 induces apoptosis and cell cycle arrest in pediatric leukemia cells at physiologically relevant concentrations. Our combination studies identified distinct anti-cancer agents that could be used for developing effective drug combination regimens with TK216. Overall, our findings provide essential preclinical data for the consideration of TK216 in early phase clinical trials for the treatment of selected high-risk and refractory childhood leukemia.

e14544 Background: PV-10 (10% rose bengal disodium; 4,5,6,7-tetrachloro-2’,4’,5’,7’-tetraiodofluorescein) is a novel therapeutic agent previously shown to have potent anti-tumor activity following intratumoral injection in melanoma and refractory neuroblastoma, and currently is undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). Given the established clinical efficacy of PV-10 in adult melanoma and hepatic cancers via intratumoral injection, there is a need to evaluate the therapeutic potential of PV-10 in high-risk and refractory adult solid tumors via systemic administration. Our study aims to identify the clinical potential of systemically-delivered PV-10 by first generating prerequisite in vitro data for adult malignancies. Methods: Cytotoxicity assays were performed using the Alamar Blue assay to study the effects of PV-10 in vitro 96-hours post-treatment against a panel of adult solid tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (HCT-116, LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues. Light microscopy and Western blotting were used to investigate apoptosis induction and target modulation in tumor cells after PV-10 treatment. Results: In vitro results from our study demonstrate that PV-10 is cytotoxic at pharmacologically relevant concentrations across the indicated cell lines. Specifically, tumor cell lines originating from testicular tissues were highly sensitive to PV-10 treatment (Mean ± SD IC50: 37.5 ± 16.4 µM; n = 3) compared to breast (117.5 ± 71.0 µM; n = 3), colorectal (64.79 µM; n = 3), and head and neck (106.6 ± 29.2 µM; n = 4) cell lines. Western blot analyses showed dose- and time-dependent activation of pro-apoptotic protein markers in caspase-3 and PARP cleavage, indicating drug-induced apoptosis. Conclusions: This study provides the first pre-clinical results of PV-10 as a novel systemically-delivered therapeutic agent for a range of high-risk and refractory adult solid tumors. Data obtained from our in vitro experiments using a broad repertoire of cell lines that represent diverse molecular and phenotypic subtypes of solid tumors in adults can serve as prerequisite pre-clinical data to establish clinical testing in these populations.

e21502 Background: Neuroblastoma (NB) is the most common extracranial solid tumor and one of the most complex and difficult to treat diseases in pediatrics. Currently, even with highly aggressive treatment protocols, the prognosis for patients with high-risk and relapsed NB remains poor. Hence, there is a clear need to identify new agents and novel therapeutic strategies for the treatment of these children. Taurolidine (TRD) is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. TRD has demonstrated anti-neoplastic activity against a range of aggressive human tumors. We present mechanistic evidence and supportive preclinical data from in vitro and animal models of refractory NB for the development of an early phase clinical trial incorporating TRD. Methods: For in vitro activity studies, a panel of cell lines derived from patients with relapsed NB (n = 6) and normal control cells were treated with increasing concentrations of TRD and cell viability was measured by alamar blue assay. Phase-contrast light microscopy, western blotting, time-lapse video microscopy and analysis of global gene expression by RNA-Seq were used to evaluate target modulation and induction of cell death pathways. Bioluminescence imaging of mice bearing NB xenografts treated with TRD was used to investigate the efficacy of TRD in vivo. Results: Cell survival data showed that TRD is cytotoxic against NB cell lines in vitro (mean IC50 value 100 µM, range 65-135 µM). Phase-contrast and time-lapse video microscopy confirmed the antitumor effects of TRD. Western blot analyses identified that TRD induced target modulation and an effective apoptotic cascade, resulting in PARP cleavage. Gene expression analyses and signaling pathway activation scores indicated alterations in the Notch, MAPK and IL-10 signaling pathways. Xenograft studies further validated the in vivo activity of TRD with decreased tumor burden in treated mice and a measurable improvement in survival. Conclusions: Our study provides key pre-clinical data on the activity and mechanism of action of TRD against NB. The findings support the rationale for further evaluation of TRD for the treatment of relapsed/refractory NB patients in an early phase clinical trial.

e22514Background: Melatonin is a natural health product currently used for the treatment of sleep disturbances and fatigue. In preliminary studies of adults with advanced cancer, melatonin at a dos...

10557Background: Children with refractory malignancies who fail conventional treatment protocols endure significant morbidity and mortality. Therefore, studies to identify tolerable and effective t...

10030 Background: Diffuse intrinsic pontine gliomas (DIPG) and high-grade astrocytomas (HGA) have dismal prognoses. We previously demonstrated in a phase 1 study that cetuximab and irinotecan was a safe and tolerable regimen. Consequently, we initiated this 2-strata phase 2 trial to investigate the safety and efficacy of weekly cetuximab given with involved field external beam radiation therapy followed by 10 cycles of cetuximab and irinotecan for DIPG and HGA as determined by the 1-year progression-free survival. Methods: Eligible patients aged 3-21 years with newly diagnosed HGA or DIPG were enrolled to parallel strata. All patients received radiation therapy (5940 cGy) with concurrent cetuximab at 250mg/m2 IV weekly for 6 weeks. Following radiation, patients received cetuximab (250mg/m2 IV) weekly and irinotecan (16mg/m2/day IV) daily x 5 for two weeks every 21 days for 30 weeks. Tumor, serum, and CSF samples were collected for correlative studies. Sera collected at the onset of rash were analyzed for inflammatory and immune-related cytokines. Results: Forty-eight patients (27 DIPG, 21 HGA) were enrolled and 45 were treated (median age 8 years; range: 3–19). Toxicities were manageable; the most common adverse events were fatigue, gastrointestinal complaints, neutropenia, rash, headache, electrolyte abnormalities, elevated ALT/AST, and fever. Grade 3-4 events in ≥10% of patients were hypokalemia and lymphopenia. 4 patients experienced cetuximab-related hypersensitivity reactions (2 grade 3 reactions). The median PFS was 9.5 months (95% CI: 7.0-12.2) for HGA and 7.8 months (7.0-8.6) for DIPG with a 1-year PFS±SE of 24±10% and 25%±10% respectively. The median OS for HGA was 17.7 months (95% CI: 14.1-18.0) and 11.5 months (8.8-14.2) for DIPG. Biological correlative studies will be presented. Conclusions: Cetuximab and radiation therapy followed by cetuximab and irinotecan is well tolerated in children. Based on the 1-year PFS, this regimen may deserve further investigation in patients with DIPG. Biological correlative studies will delineate the mechanisms of the rash and possible implications for EGFR-targeted therapeutics in such patients. Clinical trial information: NCT01012609.