Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

1317 Acute myelogenous leukemia (AML) is difficult to treat successfully in both adult and pediatric patients using conventional chemotherapy. Mutated or aberrantly expressed proteins on the cell surface of myeloblasts provide a focus for targeted therapy which 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. We report here that the Mer receptor tyrosine kinase is upregulated in approximately 80% of AML cell lines and patient samples, and explore the therapeutic potential of Mer inhibition. We assessed the prevalence of Mer expression in AML. Western blot and flow cytometric analysis demonstrated expression of Mer in greater than 85% (12/14) of AML cell lines. Mer expression was also assessed at the time of diagnosis and relapse in both pediatric and adult patient samples using flow cytometry. We found that Mer was expressed on leukemic blasts in 80% of 36 pediatric and 100% of 10 adult patients at the time of diagnosis with AML. Additionally, 100% of 11 patients expressed Mer at the time of relapse. Furthermore, when analyzing patient samples at relapse compared to the same patient's diagnostic sample, there was a trend toward increased Mer expression. This is in contrast to normal bone marrow myeloid progenitors from healthy donors, which express little or no Mer. Using two independent shRNA constructs directed against Mer, we analyzed the effects of Mer inhibition in two Mer expressing AML cell lines. Mer knock-down and control cell lines were assessed for apoptosis by flow cytometry after serum starvation and staining with Yo-Pro-1 iodide and propidium iodide. Compared to AML cell lines transduced with a non-silencing control shRNA (shControl), cell lines expressing reduced levels of Mer protein demonstrated significantly more apoptosis (p<0.05). Additionally, when these cell lines were plated in equal number in methylcellulose, cell lines with reduced Mer expression demonstrated decreased colony forming potential compared to shControl cells (p<0.01). Mer knock-down and control cell lines were injected into NOD-SCID-gamma mice after sublethal irradiation and the mice were monitored for development of leukemia. Mice injected with myeloblasts expressing decreased levels of Mer demonstrated significantly prolonged symptom-free survival compared to mice transplanted with shControl AML cells (p<0.001). To further explore the effects of Mer inhibition in AML, we used a novel small molecule tyrosine kinase inhibitor (UNC1666), which has high specificity to Mer. Three Mer expressing AML cell lines were treated with UNC1666 in vitro; treatment reduced phosphorylation of Mer and the downstream signaling molecules ERK1/2 and STAT6. Additionally, treatment with UNC1666 resulted in significant induction of apoptosis (p<0.05) by flow cytometric analysis after staining with Yo-Pro-1 iodide and propidium iodide, and dose-dependent inhibition of colony formation in soft agar, when compared to vehicle treated cells In summary, the upregulation of Mer expression in patient samples and the functional effects on survival with Mer shRNA knockdown help validate Mer as a new and attractive AML therapeutic target. Furthermore, a novel Mer tyrosine kinase inhibitor decreased myeloblast cell survival, providing evidence that Mer is a druggable target in AML. Disclosures: Kireev: WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Wang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

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

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.

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