UNC569 As Novel Small Molecule Mer Receptor Tyrosine Kinase Inhibitor for Treatment of ALL

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Pediatric cure rates for ALL have dramatically increased in recent years as intensified therapeutic regimens have been developed. However, intensified therapy is associated with a significant and increased risk of short- and long-term toxicities. In addition, patients who relapse, do not reach remission, or have certain cytogenetic abnormalities have a poor prognosis and limited treatment options. Therefore, novel and less toxic therapies are needed. Tyrosine kinases are frequently abnormally regulated in cancer cells. Mer receptor tyrosine kinase is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, dramatically increases the sensitivity of leukemia cells to cytotoxic agents, and significantly delays development of leukemia in a mouse model. Thus, Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule selective for Mer TK (UNC569) and characterization of its biochemical and anti-tumor activities in cell culture models of ALL.

UNC569 is a substituted pyrazolopyrimidine that has been developed by a structure-based design and iterative medicinal chemistry from the known Mer/C52 cocrystal structure. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed through a LabChip EZ Reader. Western blot analysis of phosphorylated and total Mer protein was used to determine Mer inhibition by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cell lines. UNC569-mediated anti-leukemia activity was determined by detection of metabolically active cells using MTT reagent after 48 hours of exposure and by determining colony-formation in methylcellulose medium in the presence of UNC569. To investigate interactions with standard ALL therapies, apoptotic and dead cells were identified by flow cytometric analysis of cells stained with YO-PRO-1 and propidium iodide dyes after treatment with a chemotherapeutic agent alone or in combination with UNC569.

UNC569 is a novel small molecule Mer TKI with potent activity against Mer kinase (IC₅₀ = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in both 697 and Jurkat ALL cells (IC₅₀ <100 nM). Reduced proliferation and/or survival of ALL cells was also observed in 697 (IC₅₀ = 0.91 ± 0.16 μM) and Jurkat (IC₅₀ = 1.55 ± 0.19 μM) cultures. Treatment with UNC569 resulted in a statistically significant, dose-dependent decrease in colony-formation in Jurkat and 697 cell lines (60 ± 13.8 % (p = 0.01) and 29 ± 12.1 % (p = 0.01), respectively, relative to untreated cultures). Treatment of 697 cells with UNC569 in combination with etoposide resulted in a statistically significant increase in apoptotic and dead cells when compared with etoposide alone (43.3 ± 4.6 % vs 33.03 ± 0.73 %, p = 0.02). Treatment of Jurkat cells with a combination of UNC569 and methotrexate also resulted in a statistically significant increase in apoptotic and dead cells relative to methotrexate alone (26.9 ± 9.5 % vs 18.5 ± 5.7 %, p = 0.03).

UNC569 is a highly effective Mer TKI that inhibits accumulation of the active phosphorylated form of Mer in B- and T-ALL cells. UNC569 mediates anti-leukemia activity against B- and T-ALL cells in culture and decreases colony-forming potential in methylcellulose. In addition, treatment with UNC569 sensitizes ALL cells to cytotoxic agents that are currently used as standard ALL therapies. Taken together, these data suggest that treatment with UNC569 may be a novel and effective ALL therapy and may be particularly effective in combination with cytotoxic therapies, thereby allowing for dose reduction and decreased incidence of toxic side effects.

No relevant conflicts of interest to declare.