Abstract
The identification of novel biologically targeted agents for the treatment of acute myeloid leukemia (AML) could potentially augment therapeutic outcome, decrease toxicity to normal tissues, and/or provide a therapy option for those who are not able to tolerate conventional therapy. Previously, Flt-3 and Mer have been identified as potential targets in the treatment of acute myeloid leukemia. Flt-3 internal tandem duplication (ITD) has been described in ∼30-40% of AML patient samples and Mer overexpression has been detected in ∼80-100% of AML patient samples. Here we describe a novel first-in-class small molecule inhibitor that has dual inhibition of these two tyrosine kinases with effective killing of myeloblasts in preclinical evaluation.
UNC1666 is an ATP competitive reversible small molecule inhibitor which potentially inhibits Mer (IC50: 0.55 nM) and Flt-3 (IC50: 0.69 nM), preventing phosphorylation of the kinase and resultant downstream signaling. In these studies, UNC1666 was analyzed in AML cell lines which were either known to be Mer-positive (Nomo-1, Kasumi-1 and U937) or positive for the Flt3-ITD (Molm-13 and MV4;11). Short term exposure of these cell lines to UNC1666 resulted in decreased AKT and STAT6 activation in a dose dependent manner compared to equivalent concentration of drug vehicle (DMSO). AML cell lines were treated with nanomolar doses of UNC1666 or equivalent concentration of vehicle for 72 hours, stained with Yo-Pro-1 iodide and propidium iodide, and were assessed for apoptosis by flow cytometry. Compared to AML cells treated with vehicle, treatment of Mer-positive cell lines with UNC1666 resulted in a two to five-fold induction of apoptosis (mean of 20±10% apoptosis after treatment with vehicle versus 66±10% after treatment with UNC1666, p<0.01). Treatment of Flt3-ITD cell lines with UNC1666 resulted in a nine-fold induction of apoptosis (mean of 10±2% apoptosis after treatment with vehicle versus 90±6% after treatment with UNC1666, p<0.001). To further evaluate for sustained effects of UNC1666 on cell growth and survival after drug removal, AML cell lines were treated with UNC1666 for 72 hours, then washed and replated in normal growth conditions to assess for possible rebound growth. Viable cell count was performed on a Cedex analyzer using trypan blue exclusion over the course of six days. Cells treated with UNC1666 continued to have a significant decrease in their ability to proliferate even after the drug was removed (day six viable cell count of 1.9 x 106 cells previously treated with vehicle versus 0.04 x 106 cells previously treated with UNC1666, p<0.01). Furthermore, when these AML cell lines were plated in equal number in soft agar, and then treated with medium containing nanomolar doses of UNC1666 for 10 days, cell lines treated with the dual inhibitor demonstrated decreased colony formation compared to cells treated with medium containing vehicle only (relative colony count of 100 with vehicle treatment versus 34±15 colonies with Mer-positive cell lines and 15±12 with Flt3-ITD cell lines after treatment with UNC1666, p<0.01).
In summary, UNC1666 is novel first-in-class small molecule with ability to inhibit kinase activity and activation of both Mer and Flt-3 tyrosine kinases. Inhibition using this molecule effectively prevented downstream activation of several intracellular signaling molecules including AKT and STAT6, induced apoptosis and decreased myeloblast proliferative capacity. These data validate dual Mer/Flt-3 inhibition as a new and attractive potentially clinically applicable therapeutic in the treatment of AML.
Disclosures:
Liu: University of North Carolina: inventor on a patent application relevant to this work, inventor on a patent application relevant to this work Patents & Royalties. Wang:University of North Carolina at Chapel Hill: This author is an inventor on patent application relevant to this work, This author is an inventor on patent application relevant to this work Patents & Royalties. Frye:University of North Carolina at Chapel Hill: This author is an inventor on a patent application relevant to this work, This author is an inventor on a patent application relevant to this work Patents & Royalties.
Targeting acute myeloid leukemia with a small molecule inhibitor of the Myb/p300 interaction