Abstract 4786: Integrated high-throughput screen to identify treatment leads for pediatric AML
Authors: Drenberg Christina D. , Shelat Anang , Orwick Shelley J. , Inaba Hiroto , Ribeiro Raul C. , Rubnitz Jeffrey E. , Gruber Tanja A. , Guy R K. , Baker Sharyn D. 
Published: July 2016

Abstract
While dramatic improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), some subtypes are associated with event free survival of &lt;40%. Given that significant improvements in long-term outcome are not expected with conventional therapy alone, new therapeutic strategies are urgently needed. Therefore, we conducted a high-throughput screen (HTS) to identify novel therapeutics for the treatment of pediatric AML. We selected a panel of 8 AML cell lines representing subtypes with the poorest prognosis. An initial primary screen was performed at a single concentration (10μM) with &gt;8000 compounds. Compounds with &gt;50% activity (N = 285) in the primary screen and known clinical candidates were prioritized for a secondary HTS performed in a dose-response manner (10 point curve, 0-10μM). Screening was performed using a 348-well plate format and a Biomek automation workstation for drug delivery. At 72 h cell viability was determined using Cell Titer Glo and automated Envision plate reader. Multiple drug classes had broad activity including: anti-malarials, cytotoxics (antimicrotubules, purine/pyrimidine antimetabolites, floxuridine, trimetrexate, topoI/II inhibitors), epigenetic modifiers (BET, DNMT and HDAC inhibitors), and agents targeting Bcl-2, Chk1, Parp, Hsp90, NF-êB, NAMPT, p53, proteasome, and Wee1. We validated 13 compounds from select drug classes (artesunate, cytarabine, gemcitabine, cabazitaxel, depsipeptide, panobinostat, vorinostat, ABT-199, RG117, bortezomib, carfilizomab, BMN673, MK-1775) using primary blast samples representing 3 high-risk groups (FLT3-ITD-positive, MLL-rearranged, FAB M7). Given the broad activity of gemcitabine and cabazitaxel across AML subtypes and the ability for repurposing in childhood AML, we further evaluated these agents alone and in combination in vitro. After 72h exposure, cabazitaxel was a potent inhibitor of viability in AML cell lines (IC50, 3-11nM) and primary blast samples (IC50, 3.4nM to &gt;10μM); gemcitabine had similar activity (IC50, 3-62nM and 102nM to &gt;10μM, respectively). The combination was evaluated using continuous and sequential administration schedules for up to 72h of treatment in several cell lines; simultaneous treatments were synergistic (CI, 0.2-0.45) and sequential treatments were additive to synergistic (CI, 0.82-0.95). Tolerable combination regimens were identified in NSG mice (5mg/kg cabazitaxel + 50mg/kg gemcitabine q3d x 4 weeks or q4d x 3 weeks) and efficacy studies in AML xenografts and primagrafts are ongoing. Further studies will aim to determine the underlying mechanisms of AML sensitivity to gemcitabine, cabazitaxel, and the combination. Our comprehensive approach to identify treatment leads provide advances in understanding the biology of a heterogeneous disease and development of novel treatment strategies with potential clinical benefit for pediatric AML.
Citation Format: Christina D. Drenberg, Anang Shelat, Shelley J. Orwick, Hiroto Inaba, Raul C. Ribeiro, Jeffrey E. Rubnitz, Tanja A. Gruber, R K. Guy, Sharyn D. Baker. Integrated high-throughput screen to identify treatment leads for pediatric AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4786.