Mer Receptor Tyrosine Kinase Is a Novel Therapeutic Target In Multiple Myeloma
Authors: Christoph Sandra , Maag Silke , DeRyckere Deborah , Graham Douglas K. , Frye Stephen V , Earp H. Shelton , Liu Jing , Yang Chao , Zhang Weihe , Wang Xiaodong , Elmaagacli Ahmet H. , Beelen Dietrich 
Published: October 2019

Abstract

Introduction
Although many novel therapeutic agents have been explored, multiple myeloma (MM) remains an incurable hematopoietic malignancy. Mer receptor tyrosine kinase (Mer TK) is a member of the TAM (Tyro3, Axl and Mer) family and is involved in the progression of several human malignancies. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity in vitro, and delays tumor progression in vivo. These observations make Mer TK inhibitors to excellent candidates for targeted therapies. We show here for the first time that Mer TK is ectopically expressed in MM cells and test the response of MM cell lines to our previously described small molecule Mer inhibitor, UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biologic effects of treatment with UNC1062, which suggests efficacy of Mer inhibitors as a novel strategy for the treatment of MM. Methods: Western blot analysis was used to determine expression of the TAM receptors and to evaluate inhibition of Mer TK activation and downstream signaling by UNC1062 in MM cell lines. UNC1062-mediated anti-myeloma activity with or without bortezomib was determined using short-term (MTT) and long-term (colony-formation) assays. Cleaved PARP and caspase 3 were detected by western blot analysis as indicators of apoptosis. Results: Western blot analysis of protein extract from MM cell lines showed that four (RPMI8226, U226, AMO-1 and KMS-12BM) out of six lines (66.6%) express Mer TK protein at varying levels, with AMO-1 expressing the least and U226 expressing the most. The MM cell lines OPM-2 and NCI-H929 did not express Mer TK. UNC1062 potently inhibits Mer TK activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through ERK and Stat5 in U226 cells. UNC1062 also reduced proliferation and/or survival in Mer TK-expressing RPMI8226 cells (IC50 = 0.98 ± 0.14 μM, n=7) and U226 (IC50 = 0.28 ± 0.09 μM, n=3) cells. Treatment with UNC1062 did not lead to a meaningful reduction of metabolically active cells in Mer-negative NCI-H929 and OPM-2 cells. In contrast, UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase 3 and cleaved PARP confirming apoptosis induction. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM-2 cells. Treatment of U226 cells with UNC1062 (800 nM) in combination with bortezomib resulted in a statistically significant reduction in relative cell number compared with bortezomib alone (0.61 ± 0.06 vs 0.41 ± 0.06, p = 0.04, n =3). Conclusion: Mer TK is ectopically expressed in the majority of investigated MM cell lines. UNC1062 treatment of Mer TK positive MM cells inhibits Mer TK activity and downstream signaling, mediates anti-neoplastic activity in liquid culture, and decreases colony-formation in methylcellulose. In addition, UNC1062 treatment sensitizes MM cells to bortezomib. Taken together, these data support further development of Mer TK inhibitors as novel MM therapy alone and in combination with bortezomib.

Disclosures:
No relevant conflicts of interest to declare.