Flt3/ITD Blocks Myeloid Differentiation Of Hematopoietic Cells By Up-Regulating Runx1

Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD.

A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD⁺AML cells (N=78) than in FLT3/ITD^-AML cells (N=190, P<0.05). The mRNA microarray analysis consistently demonstrated that Runx1 is up-regulated by Flt3/ITD in Ba/F3 cells. Up-regulation of Runx1 by Flt3/ITD was validated in Ba/F3 cells and 32D cells by quantitative RT-PCR. Incubation of control 32D cells with 20 ng/ml of G-CSF increased the number of Gr-1⁺/Mac-1⁺cells, whereas the induction of myeloid differentiation by G-CSF was abrogated by the overexpression of Flt3/ITD in 32D cells. By contrast, transduction of shRNA specific for Runx1 into Flt3/ITD⁺32D cells inhibited the expression of Runx1 mRNA by 60 % but increased the number and the proportion of Gr-1⁺/Mac-1⁺cells ; these effects were enhanced by incubation with G-CSF. These data indicate that Runx1 mediates the block of differentiation toward the myeloid lineage that is induced by Flt3/ITD. Moreover, the number of colony-forming units (CFU) over-expressing Flt3/ITD cultured in the absence of growth factors was reduced by Runx1-shRNA without affecting the total cell number in the suspension culture, as compared to Flt3/ITD⁺32D cells transduced with control-shRNA. This implies that antagonizing Runx1 facilitates the production of terminally differentiated cells that have lost colony-forming ability, thereby reducing the CFU number without altering the total number of cells. Finally, Runx1-shRNA inhibited the formation of secondary CFU colonies derived from the primary Flt3-ITD-over-expressing CFU colonies.

Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD⁺cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD⁺cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD⁺cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD⁺hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD⁺AML cells in AML patients, in addition to inhibiting their aberrant proliferation.