Mer Receptor Tyrosine Kinase Is A Potential Therapeutic Target in Acute Myeloid Leukemia

Abstract 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.