Abstract
Signal transducer and activator of transcription (STAT) family proteins play crucial roles in the cytokine signaling pathways which regulate survival and proliferation of normal hematopoietic cells. However, the role of STAT proteins in regulating survival in leukemia remains poorly defined. STAT3 and STAT5, for example, have been reported to be constitutively activated in acute myeloid leukemia (AML) cells, however the physiologic significance of this activation is unknown. In order to better understand the role of STAT3 and STAT5 in AML biology, we studied their expression, activation, and requirement for cell growth in several AML cell lines and primary AML cells collected from patients at the University of Pennsylvania Cancer Center. We first confirmed the activation of STAT3 and STAT5 in primary AML cells by western blotting. An analysis of AML patient samples revealed elevated levels of constitutive STAT3 phosphorylation in 6 of 7 patient samples and constitutive STAT5 phosphorylation in 8 of 9 patient samples. In addition, 6 AML cell lines (K562, HL-60, MOLM-14, U937, KG-1, NB4) displayed constitutive STAT3 and STAT5 activation. In order to evaluate the functional significance of constitutive activation of STAT3 and STAT5 in AML cells, we designed and synthesized short interfering RNAs (siRNAs) to silence the expression of these proteins. For initial characterization, the siRNAs were delivered to MOLM-14 cells using an AMAXA nucleofector device (AMAXA, Inc. Gaithersburg, MD)(Program O-17/Solution V). Nucleofected siRNA diminished STAT3 expression by 85% at 24 hour but had little effect on cell proliferation (13%±3% decrease at 24 hour, 8%±4% at 48 hour, 5%±1% at 72 hour) compared to control siRNA treated cells. In contrast, STAT5 siRNA decreased STAT5 expression 80% at 24 hour compared to control treated cells but inhibited cell proliferation by 19%±1% at 24 hour, 22%±1% at 48 hour, 16%±3% at 72 hour in comparison to control siRNA treated cells suggesting a more important role for STAT5 in regulating cell proliferation. To study the effect of these siRNA molecules in primary AML cells, we first determined our ability to nucleofect primary cells by examining delivery efficiency of fluorescein labeled siRNA. Four different patient samples were evaluated and the mean ± SD of cells successfully transfected was 52%±12. Transfection of multiple AML patient samples with STAT3 siRNA decreased STAT3 expression but led to only modest decrease (6–18% at 48 hour, 7–36% at 72 hour) in AML cell survival. However, transfection of cells with STAT5 siRNA, but not control siRNA, led to a consistent decrease (25–54% at 48 hour, 21–60% at 72 hour) in AML cell survival. The decrease in survival was proportional to the transfection efficiency in the different samples. These results provide the first evidence that STAT5 expression and activation is necessary for the survival of primary AML cells. In fact, the data suggests a greater role of STAT5 in survival of primary cells than in survival of AML cell lines. Accordingly, STAT5 appears to be a legitimate target for the treatment of AML.
Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia