Flt3/ITD Enhances Hematopoietic Cell Migration Towards Chemokine CCL2 Independent of Blocking the Negative Feedback Mechanism on Rho-Associated Kinase

Internal tandem duplication mutations in the Flt3 gene (Flt3/ITD) found in patients with AML is associated with extremely poor prognosis. Our previous report demonstrating Flt3/ITD-mediated enhancement of hematopoietic cell migration towards chemokine CXCL12 (SDF1) suggests that Flt3/ITD likely facilitates dissemination of AML cells in the patients. Following studies showed that CXCL12 transiently up-regulated the expression of Rho-associated kinase-1 (Rock1) but subsequently down-regulated Rock1 expression in the control cells, whereas this effect was abolished by Flt3/ITD. The results demonstrated that CXCL12 generates negative regulatory feedback on Rock1 expression to prevent excessive migration to CXCL12 in the control cells, whereas this mechanism is abrogated by Flt3/ITD, thereby inducing deregulated cell migration (Onishi et al. ASH 2012). However, it is not known if Flt3/ITD augments migration to other chemokines and whether Flt3/ITD-induced blockage of the negative feedback loop on Rock1 expression reflects a specific effect on CXCL12/CXCR4 signaling pathway or more global change by ITD-Flt3. We found that mRNA for CCR2, a receptor for chemokine CCL2, is expressed in human AML cells. Herein, we investigated the effect of Flt3/ITD on migration to CCL2 in hematopoietic cells.

Expression of CCR2 was significantly higher in the M4 and M5 cases with AML compared to other FAB subtypes that are deposited in the public gene expression database. However, there was no difference in the mRNA level for CCR2 between Flt3/ITD+ and Flt3/ITD^neg cases. Consistent with AML samples, expression of surface CCR2 protein in Ba/F3 cells transfected with Flt3/ITD is equivalent to the control cells lacking Flt3/ITD. While the control Flt3/ITD^neg Ba/F3 cells failed to migrate towards 1, 5, 10 and 50ng/mL of CCL2, Flt3/ITD marginally but significantly enhanced the cell migration towards 5ng/mL of CCL2 in Ba/F3 cells within 4 hours compared to control. In the Flt3/ITD^neg cells exposed to 5ng/mL of CCL2, the mRNA expression of Rock1 continued to increase without being down-regulated to the basal level within 4 hours, and did not show any biphasic changes. In the Flt3/ITD⁺ Ba/F3 cells, however, Rock1 expression was significantly elevated compared to Flt3/ITD^neg cells prior to incubation with CCL2, but down-regulated to 50% of the original level by 5ng/mL of CCL2 within 30 minutes. In contrast, Rock1 expression was barely affected and remained elevated by CXCL12 in Flt3/ITD⁺ Ba/F3 cells.

Elevated expression of CCR2 in the M4 and M5 AML suggests that CCR2 signaling pathways can regulate migration of AML cells with monocytic lineage. Similar to CXCL12, migration towards CCL2 was also enhanced by Flt3/ITD without up-regulating CCR2 expression, suggesting that the enhanced chemotaxis by Flt3/ITD is not specific to CXCL12 and likely attributed to qualitative changes in the CCL2/CCR2 signaling pathway rather than their quantitative increase. Down-regulation of Rock1 expression by CCL2 in Flt3/ITD⁺ Ba/F3 cells may represent one of the qualitative changes in the CCL2 signaling pathway. However, blocking the CXCL12-induced negative regulatory mechanism on Rock1 expression existing in the Flt3/ITD⁺ cells was not identified in the CCL2 signaling. These data indicate that while enhancement in cell migration to chemokines by Flt3/ITD is not specific to CXCL12, blocking the negative feedback mechanism on Rock1 expression is not necessarily used in other chemokine signaling pathways. Our data suggests that Flt3/ITD mutations regulate trafficking of AML cells by modulating various chemokine signaling, but divergent molecular mechanism is involved in regulating cell migration towards different chmeokines.