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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.

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.

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro evaluations of drug response.This methodology was combined with a previously validated molecular pathway analysis program to identify effective agents or combinations for a subsequent informed precision clinical trial. Methods: Gene expression profiles from refractory pediatric leukemic cells were analyzed against similar data from more than 12, 000 tumors and outlier analyses were carried out to generate a list of overexpressed genes. This information was computed to identify hypothetically activated pathways, druggable targets and potential agents from a panel of FDA approved drugs. A bone marrow stromal cell line was established and characterized that has been shown to support leukemia cell proliferation in vitro. Briefly, stromal cells were co-cultured with leukemic cells at pre-determined ratios with and without the drugs identified in the genomic analysis. After four days in culture, leukemic cells were re-suspended and analyzed for proliferation. Target modulation and activated cell death pathways were queried by Western blot analyses. Results: Multiple targets and potential agents for effective therapeutics were identified against an initial set of relapsed leukemia specimens. For example, in patient # P700491 (pre B-ALL) gene expression data sets revealed clustering within the area of ALL and AML in the reference cancer genomics data. Comparative tumor RNA seq outlier analysis showed molecular abnormalities in BTK, JAK3 and PIK3CD, corresponding to molecular categories of RTK, JAK-SAT and PI3K-AKT-mTOR pathways targetable by the drugs Ibrutinib, WHI-P131 and Idelalisib, respectively. However, in vitro studies showed significant cell killing with Idelalisib and not with the other two agents. Target modulation assays showed effective induction of apoptosis including PARP cleavage in Idelalisib treated leukemia cells compared to controls, indicating the feasibility of this approach to effectively identify potentially applicable agents for this individual patient. Conclusions: We demonstrate the ability of a newly cloned bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro target modulation and target validation analyses for cytotoxicity. This methodology was combined with a previously demonstrated molecular pathway interrogation program to ascertain effective agents or combinations for an experimentally informed precision clinical trial. Importantly, our data showed that genomically identified actionable targets are not universally predictive of tumor response and an in vitro cytotoxicity analysis step may enhance the accuracy of this approach. We describe the practical advantages and versatility of this work-flow to inform the selection of agents in future umbrella trials. It is anticipated that the information obtained will lead to an applicable clinical trial the near future. Disclosures Narendran: Bayer: Honoraria, Other: CANTRK Advisory Board .