Figure 4. Syk inhibition affects a diverse set of AML cell lines.

(A) Western immunoblot of AML cell lines reveals MOLM-14 and KG-1 cell lines to be highly phosphorylated at the p-Syk (Y525/526) enzymatic site.

(B) MOLM-14 and KG-1 cells were treated with vehicle or R406 for 6 days. Cells were stained with annexin V-FITC and propidium iodine (PI) and evaluated by flow cytometry. R406 treatment induced increased annexin V positive cells in a dose responsive manner consistent with apoptosis.

(C) MOLM-14 and KG-1 cells were infected with shRNAs targeting SYK and luciferase. After 8 days of infection, cell viability was evaluated at days 0, 3, and 6 using an ATP-based assay. Genetic loss of Syk resulted in dramatic decrease in proliferation. Error bars depict mean ±SD across ratios of 6 replicate measurements at each time point relative to the 6 replicate measurements at time zero.

(D) Ectopic expression of TEL-Syk immune to the SYK-directed shRNA Syk_1 in MOLM-14 cells rescues the effects of Syk knockdown on cell viability. Cell viability was evaluated 4 days after infection at days 0, 3, and 6 with an ATP-based assay. Error bars depict mean ±SD across ratios of 8 replicate measurements at each time point relative to the 8 replicates at time zero.

(E) May Grunwald Giemsa staining of KG-1 cells 12 days post infection with shRNAs targeting SYK versus a luciferase shRNA control demonstrate evidence of macrophage-like differentiation. Images were acquired with an Olympus BX41 microscope, 1000X magnification under oil, and Qcapture software. The scale bar equals 25 μm.

(F) KG-1 cells were treated with R406 versus DMSO and the 32-gene differentiation signature measured at 24 hours. R406 induces a myeloid differentiation signature as measured by a Weighted Summed Score (Differentiation Score). Error bars depict mean ±SD across 8 replicates.