Evaluation of UNC569, a Novel Small Molecule Mer Inhibitor for the Treatment of ALL in Vitro and in Vivo.

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Current chemotherapeutic regimens are associated with short- and long-term toxicities. Therefore, novel, less toxic therapies are needed. Mer receptor tyrosine kinase (RTK) is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity, and delays development of leukemia in vivo. Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule inhibitor for Mer RTK (UNC569) and demonstrate efficacy as a novel strategy in the treatment of ALL.

UNC569 is a substituted pyrazolopyrimidine. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis assay. Western blot analysis was used to determine inhibition of phospho-Mer and downstream signaling by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cells. UNC569-mediated anti-leukemia activity was determined in short- (MTT) and long-term (colony-formation) assays. Diagnostic bone marrow or peripheral blood samples were obtained at the Children's Hospital Colorado. We established a luciferase expressing ALL xenograft model in NOD scid gamma (NSG) mice to evaluate the effects of UNC569 on leukemia progression. NSG mice were transplanted with luciferase-tagged 697 cells and treated for three weeks with an orally bioavailable UNC569 formulation (15 mg/kg body weight, qd). Response was monitored twice weekly using the IVIS200 Imaging System. To examine effects of UNC569 on leukemia progression in a second model, we used transgenic zebrafish ectopically expressing human MYC. These fish develop T-cell malignancy at high penetrance. Cancers in these fish are labeled by T-lymphocyte-specific enhanced green fluorescent protein (EGFP), allowing measurement of treatment responses. Leukemic adult zebrafish were treated over three weeks with 3 μM or over 2 weeks with 4 μM UNC569. Tumor responses were monitored weekly using an Olympus MVX10 Imaging System.

UNC569 is a novel Mer TKI with potent activity against Mer RTK (IC₅₀ = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in ALL cell lines (697 IC₅₀ = 141±15 nM, Jurkat IC₅₀ = 193±56 nM). Treatment with UNC569 resulted in inhibition of phosphorylation of Erk1/2 and Akt. Reduced proliferation/survival was observed in ALL cells treated with UNC569 (697 IC₅₀ = 0.5±0.1 μM, Jurkat IC₅₀ = 1.2±0.2 μM). Treatment with UNC569 also resulted in a statistically significant, dose-dependent decrease in colony-formation compared to control cultures in Jurkat (100.1±23.4 vs 25.6±6.4 colonies, p=0.04, n=3) and 697 cells (95.9±16.8 vs 14.8±12.8 colonies, p=0.02, n=3). Similarly, treatment with UNC569 reduced colony formation in methylcellulose compared to control cultures in one of three Mer RTK positive primary ALL patient samples (270.1±18.9 vs 134.0±6.4 colonies). No significant reduction of colony formation was observed in five Mer negative primary ALL patient samples. NSG mice transplanted with luciferase-expressing 697 B-ALL cells and treated with UNC569 (15 mg/kg/d) had significantly decreased leukemia burden compared to vehicle-treated control mice as measured by bioluminescence imaging (86.1×10⁶±19.2×10⁶ photons/second (n=15) vs 29.6×10⁶±9.0×10⁶ photons/second (n=10), p=0.04). Similarly, in the zebrafish T-ALL model, responses to UNC569 treatment (defined as >25% decrease in disease burden) were observed in 84% of animals treated with 4 μM UNC569 for 14 days (n = 16/19) and 77% of animals treated with 3 μM UNC569 for 21 days (n = 7/9).

UNC569 is an effective Mer TKI that inhibits activation of Mer in ALL cells, mediates anti-leukemia activity against ALL cells in culture, and decreases colony-formation in methylcellulose. In addition, UNC569 has an anti-leukemia effect in primary ALL patient samples that express Mer protein, in a murine xenograft B-ALL model, and in a transgenic zebrafish model. Taken together, these data support further development of Mer TKI as a novel and effective ALL therapy.

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. 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. Yang: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.