The Internal-Tandem-Duplication of Flt3 Mutations Augment Chemotaxis in Hematopoietic Cells by Blocking the Down-Regulation of Rho-Associated Kinase That Is Induced by SDF1/CXCR4 Signaling
Authors: Onishi Chie , Mori Satomi , Hirade Tomohiro , Abe Mariko , Yamaguchi Seiji , Kapur Reuben , Fukuda Seiji 
Published: October 2019

Abstract
Abstract 1319
Internal Tandem Duplication mutations of the Flt3 gene (ITD-Flt3) and the deregulation of CXCR4 expression in patients with AML are associated with a poor prognosis (Rombouts et al. Blood 2004). ITD-Flt3 induces myeloproliferative disease with extramedullary infiltration in transplantation models, thereby suggesting that ITD-Flt3 can regulate the dissemination of leukemia cells. We have reported that ITD-Flt3 mutations enhance the migration of mouse hematopoietic cells toward SDF1 and modulate their homing in vivo (Fukuda et al. Blood 2005, Exp Hematology 2006). However, the molecular mechanism responsible for the aberrant migration of ITD-Flt3+ cells toward SDF1 remains unknown. A recent report has demonstrated that Rho-Associated Kinase (Rock) regulates the proliferation of ITD-Flt3+ hematopoietic cells (Mali et al. Cancer Cell 2012). In this report, we investigate the functional role of Rock1 in the aberrant hematopoietic cell migration induced by ITD-Flt3.
The migration of Ba/F3 cells containing 3 different ITD-Flt3 variants (N51, N73, N78: ITD-Flt3+ cells) toward SDF1 (0–500 ng/ml) was significantly increased compared with Ba/F3 cells lacking ITD-Flt3 (wild-type Flt3+ cells; P<0.01, N=4). Moreover, the enhanced migration of the ITD-Flt3+ cells toward SDF1 was reduced with the treatment of the ITD-Flt3 inhibitor, CEP701, and the CXCR4 antagonist, AMD3100. CXCR4 expression levels in all ITD-Flt3+ cells were reduced compared with the wild-type Flt3+ cells (50±6% reduction), thereby indicating that the enhanced migration of the ITD-Flt3+ cells toward SDF1 is independent of CXCR4. The incubation of wild-type Flt3+ cells with the Rock inhibitors, Y27632 and H1152, reduced the migration toward SDF1 in a dose-dependent manner. Similarly, the shRNA specific for Rock1 significantly down-regulated the migration of the wild-type Flt3+ cells toward SDF1 (72±6% reduction, N=3, P<0.01). The addition of the Flt3-ligand (FL) toward SDF1 accentuated the migration of the wild-type Flt3+ cells compared with SDF1 alone, which was also inhibited by H1152. These data indicate that Rock activity is necessary for the cell migration toward SDF1 in the presence or absence of Flt3 signaling. However, Rock1 mRNA was down-regulated by treatment with 100ng/ml SDF1 in the wild-type Flt3+ cells (52±8% reduction, N=6, P<0.001). In contrast, the FL up-regulated Rock1 mRNA expression in wild-type Flt3+ cells in a dose-dependent manner (N=3, P<0.05). Similarly, ITD-Flt3 mutations enhanced Rock1 mRNA expression (2.1±0.5-, 2.2±0.2-, and 3.2±0.6-fold increases in N51, N73 and N78, respectively; P<0.05). The reduction of Rock1 mRNA induced by SDF1 in the wild-type Flt3+ cells was abrogated by the treatment with 10ng/ml of FL. Moreover, the addition of 100 ng/ml of FL to SDF1 enhanced Rock1 expression compared with the vehicle alone in the wild-type Flt3+ cells (55±19% increase, N=3, P<0.01), thereby indicating that Flt3 signaling antagonizes the SDF1-induced down-regulation of Rock1 expression. Similarly, Rock1 levels in the 3 different ITD-Flt3+ cells that migrated toward 100ng/ml of SDF1 were significantly higher compared with the wild-type Flt3+ cells that migrated toward SDF1 (2.2±0.5-, 4.2±1.1- and 1.4±0.2-fold increases in N51, N73 and N78, respectively; P<0.02). The incubation of the ITD-Flt3+ cells with Y27632 and H1152 lead to a reduction in the enhanced migration toward SDF1 in a dose-dependent manner. The shRNA specific for Rock1 significantly down-regulated the migration of the ITD-Flt3+ cells toward SDF1 (72±8% reduction, P<0.02), thereby indicating that Rock1 supports the enhanced cell migration toward SDF1 that is induced by ITD-Flt3. Although SDF1 enhanced the phosphorylation of MYPT1, a downstream substrate of Rock1, in both the wild-type Flt3+ and ITD-Flt3+ cells, the phosphorylation levels remained elevated until 4 hours in ITD-Flt3+ cells compared with the wild-type Flt3+ cells, in which the signal faded away earlier.
Our data demonstrates that SDF1 down-regulates Rock1 expression in the absence of Flt3 signaling, while ITD-Flt3 mutations augment cell migration toward SDF1 by blocking the down-regulation of Rock1 that is induced by SDF1 signaling. Manipulating the SDF1/Rock1 axis that is modulated by ITD-Flt3 may be clinically beneficial for antagonizing the aberrant dissemination of AML cells in patients.

Disclosures:
No relevant conflicts of interest to declare.