Knockdown of the Nuclear Oncogene SKI Inhibits Flt3-ITD Induced Signaling in 32D - Flt3-ITD Cells.

Abstract

Ski is a nuclear oncogene implicated in repressing TGF-β signaling. Recently, we found that Ski is overexpressed in AML, especially in AML with deletion of chromosome 7 or 7q−. We also showed that Ski inhibits myeloid differentiation by preventing retinoic acid signaling in U937 cells. It is unclear, however, how Ski exerts its oncogenic effect in myeloid leukemia cells. In order to address this question, we took advantage of the factor-independent murine myeloid progenitor cell line 32D, which is rendered factor independent via expression of Flt3 internal tandem duplication (Flt-3-ITD). We found that Ski protein expression is upregulated in Flt3-ITD transfected 32D cells (32D-Flt3) when compared to vector transfected 32D control cells (32D-V). Ski upregulation is reversed by treatment with specific Flt3-ITD inhibitor PKC412. As Flt3-ITD transformation is known to mediate factor-independent activation of STAT5 we investigated STAT5 phosphorylation status in these cells after treatment with PKC412 or knockdown of Ski with specific siRNA targeted to murine Ski (mSki). As expected, expression of phosphorylated STAT5 (pSTAT5) as measured by Western blot and flow cytometry was strongly reduced after PKC412 treatment. Interestingly, a similar reduction of pSTAT5 expression was also achieved by siRNA treatment against mSki but was not seen in non-silencing siRNA treated 32D-Flt3 cells. In parallel, transfection of siRNA against mSki, but not of non-silencing RNA led to significant inhibition of cell proliferation in 32D-Flt3-ITD cells and to a much lesser extent in 32D-V- cells. Together, these data suggest that in in addition to AML with −7 / del7q, Ski may also be implicated in AML with Flt3-ITD, as it regulates Flt3-ITD induced STAT5 phosphorylation, which itself is a key transcription factor in leukemogenesis.