Abstract
Abstract 938
KIT is a receptor tyrosine kinase (RTK) and its aberrant activities resulting from protein overexpression and/or activating mutations are associated with a number of malignancies including core binding factor (CBF) AML [e.g., patients with t(8;21) or inv(16) or molecular equivalent RUNX1/RUNX1T1 or CBFB/MYH11, respectively]. RTK inhibitors (e.g. PKC412) have been shown to suppress aberrant KIT activity and delay tumor growth, but they are active only on distinct types of KIT mutations (KITmut). Furthermore, resistance to these inhibitors, as a result of secondary mutations or KIT overexpression, is emerging. Thus, we hypothesize that direct inhibition of KIT gene transcription may be a valuable therapeutic approach to override aberrant KIT expression and activity. Here, we described the regulatory and functional role of Sp1/NFkB-miR29b feedback loop in KIT-driven leukemia that can be targeted pharmacologically. Applying chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) to RUNX1/RUNX1T1-positive Kasumi-1 cells, we demonstrated that while, the Sp1/NFkB complex was enriched on KIT promoter and acted as gene transactivator thereby leading to KIT overexpression, Sp1/NFkB recruited HDAC1 and HDAC3 to miR29b regulatory region thereby epigenetically repressing miR29b. This microRNA, when expressed, targeted Sp1 and eventually decreased Sp1/NFkB-mediated gene transactivation, including that of KIT. In agreement with these, we showed that when Sp1, NFkB and HDAC1 were transiently overexpressed in Kasumi-1 cells, increased KIT expression and decreased miR29b transcription occurred. In contrast, siRNA knockdown of Sp1, NFkB and HDAC1 augmented miR29b level and decreased KIT transcription. Moreover, ectopic miR29b expression impaired Sp1/NFkB repressor complex on the promoter of endogenous miR29b, thereby resulting in re-expression of the endogenous microRNA and further inhibition of Sp1/NFkB-dependent KIT transcription. Importantly, the activity of Sp1/NFkB/HDACs complex can also be pharmacologically modulated leading to restored miR29b transcription and abrogated KIT expression. We showed that pharmacologic interference with Sp1/NFkB/HDACs using their respective inhibitors, such as bortezomib (0, 6, 20, 60 and 100nM for Sp1 and NFkB), mithramycin A (150 and 300ng/ml for Sp1), bay 11-7082 (3μM for NFkB) and OSU-HDAC42 (1μM for HDAC), upregulated miR29b at early time point (6 hours) and decreased Sp1 and in turn KIT expression in KIT overexpression cell lines (e.g., Kasumi-1) and AML patient blasts. EMSA and ChIP assay demonstrated that bortezomib or HDAC42-mediated KIT repression and miR29b upregulation occurred through the dissociation of Sp1/NFkB complex from the corresponding promoter. To further investigate the therapeutic potential of targeting KIT over-expression in leukemia, we stably expressed KIT wild type (KITwt) or KITmut (D816V) in FDC-P1 cell line (murine non-tumorigenic cells derived from myeloid precursors), and we evidenced that both KITwt and KITmut promoted cell proliferation that was overcome by bortezomib in clonogenic assay. In in vivo study, when NOD/SCID mice were engrafted with FDC-P1/KITmut cells (5×106/mouse), they developed significant splenomegaly and marrow blast infiltration through KIT overexpression. When leukemia-carrying mice were treated with bortezomib (1mg/kg) for 48 hours, we observed an obvious increase of endogenous miR29b expression and a significant reduction of KIT expression. Leukemic mice that received 1mg/kg of bortezomib twice/week for 3 weeks starting on day 21 after engraftment (n=5 mice/group) showed no evidence of splenomegaly and had a significantly longer median survival [59 days (twice/week) vs 28 days (vehicle-treated), p=0.0036], compared to vehicle-treated mice that instead showed massive splenomegaly. Cytospin of marrow and histopathology of spleen and liver showed that vehicle-treated mice displayed extensive blast infiltration that was instead absent in bortezomib-treated mice. Altogether, our study revealed a previously unrecognized protein-microRNA regulatory network whose imbalance contributes to KIT-driven leukemia. As the aberrant activity of this network is pharmacologically targetable, this discovery may be quickly translated into the clinic as a novel therapeutic approach for KIT-driven AML and other malignancies.
Disclosures:
No relevant conflicts of interest to declare.
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