Abstract 5524: Library screen to rapidly determine activity against normal and mutant bone marrow progenitor cells.
Authors: Hong Wan Xing , Akutagawa Jon , Chen Steven , Arkin Michelle , Braun Benjamin S. 
Published: August 2014

Aberrant signal transduction plays a central role in the pathogenesis of myelodysplastic syndrome/ myeloproliferative neoplasm (MDS/MPN), as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the key role of Ras signaling in hematopoietic neoplasms, there are currently no signal transduction inhibitors with established efficacy. This necessitates a screen of inhibitors that may potentially reveal novel therapeutic strategies and inform efforts to treat neoplasms driven by hyperactive Ras signaling.

Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 (n= 10) and wildtype (WT) mice (n=18) were utilized in the screening of 94 different inhibitors. A disease relevant, homogenous population of PreGM cells identified as Lineage lo/- Sca1- c-kit+ CD34+ FcγRII/III- CD105- CD150- was purified from bone marrow using flow cytometry. The PreGM cells were then sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1E-7× (5E-7ug/ml) to 1× (5ug/ml). DMSO and cytotoxic agents (Cytarabine, Adriamycin, Gemcitabine) served as negative and positive controls respectively. The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc’ package from the R Project for Statistical Computing.

The 94 drug candidates tested in this screen included cytotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. However, none of the compounds tested were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells when compared to WT cells. Furthermore, the IC50s calculated for mutant and WT cells were not significantly different across all inhibitors tested.

The majority of compounds tested were either FDA approved drugs or agents used in recent clinical trials. Hyperactive Kras signaling is a major underlying etiology for MDS/MPN, and thus we designed our study to test the efficacy of these candidates against mutant Kras cells. Our results indicate that mutant cells have similar drug sensitivities as normal cells over a broad range of mechanistic approaches. This leads us to believe that "synthetic" lethal opportunities may not be the approach of choice for treating Kras driven neoplasms. However, these findings have directed our efforts towards compounds that target upstream mediators of Kras signaling and future endeavors are currently underway to use the assay system to assess the activity of a highly curated library of kinase inhibitors on mutant and WT proliferation.

Citation Format: Wan Xing Hong, Jon Akutagawa, Steven Chen, Michelle Arkin, Benjamin S. Braun. Library screen to rapidly determine activity against normal and mutant bone marrow progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2013-5524