Abstract 1743: A dual FLT-3 and MER tyrosine kinase small molecule inhibitor in acute myeloid leukemia cell lines and patient samples
Authors: Sherick Alisa Lee , Menachof Kelly , Hill Amanda , Rinella Sean , DeRyckere Deborah , Liu Jing , Wang Xiaodong , Frye Stephen , Earp H. Shelton , Graham Douglas 
Published: May 2015

Abstract
FLT-3 and MER tyrosine kinases have been previously identified as potential targets in the treatment of acute myeloid leukemia (AML). Expression of FLT-3 internal tandem duplication (ITD) occurs in ∼30-40% of AML patient samples and MER overexpression has been detected in ∼80-100%. Here we describe a novel first-in-class small molecule inhibitor that has potent activity against both of these kinases and mediates growth inhibition or apoptosis of cell lines and patient myeloblasts.
UNC1666 is an ATP-competitive reversible small molecule inhibitor that potently inhibits FLT-3 and MER, preventing phosphorylation of these kinases and resultant downstream signaling. In these studies, the effects of treatment with UNC1666 were analyzed in FLT3-ITD-positive (Molm-13 and MV4;11) and MER-positive (Kasumi-1 and U937) AML cell lines and in primary AML patient samples with variable expression of FLT3-ITD and MER. Short term exposure to UNC1666 in cell lines that express either a FLT3-ITD or MER resulted in a dose-dependent decrease in AKT and STAT6 activation compared to cells treated with vehicle, confirming that UNC1666 inhibits both targets in cell-based assays. AML cell lines were also stained with Yo-Pro-1 iodide and propidium iodide and analyzed by flow cytometry to determine induction of apoptosis in response to treatment with UNC1666. Treatment of MER-positive cell lines with UNC1666 resulted in a two to five-fold induction of apoptosis relative to vehicle-treated cells (66±10% and 20±10% apoptotic cells respectively; p&lt;0.01). Treatment of FLT3-ITD cell lines with UNC1666 resulted in an even more dramatic nine-fold induction of apoptosis (90±6% verses10±2% in vehicle-treated cultures, p&lt;0.001). When AML cell lines were cultured in soft agar, treatment with the dual inhibitor resulted in decreased colony formation compared to cells treated with vehicle (relative colony counts were 100 for vehicle-treated cultures versus, 34±15 for MER-positive cell lines and 15±12 for FLT3-ITD cell lines treated with UNC1666, p&lt;0.01). Six primary patient samples that were MER and/or FLT3-ITD positive were analyzed in similar assays and exhibited dose-dependent induction of apoptosis and near complete inhibition of colony formation in methylcellulose after treatment with UNC1666.
In summary, UNC1666 is a novel first-in-class small molecule with ability to inhibit activation of both FLT-3 and MER tyrosine kinases. Treatment with UNC1666 blocked activation of pro-survival signaling, including AKT and STAT6, induced apoptosis, and decreased myeloblast colony-forming potential in FLT3-ITD positive and MER-expressing AML cell lines. Moreover, similar anti-leukemia effects were observed in primary samples from patients with AML. These data validate dual FLT-3/MER inhibition as a new and attractive approach for treatment of AML with potential for clinical application.
Citation Format: Alisa Lee Sherick, Kelly Menachof, Amanda Hill, Sean Rinella, Deborah DeRyckere, Jing Liu, Xiaodong Wang, Stephen Frye, H. Shelton Earp, Douglas Graham. A dual FLT-3 and MER tyrosine kinase small molecule inhibitor in acute myeloid leukemia cell lines and patient samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2014-1743