Abstract
Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.
Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c+ acute myeloid leukemia cells in vitro
