ITD-Flt3 Enhances Migration of Hematopoietic Cells through Aberrant CXCR4 Signaling Pathways Overlapping but Functionally Distinct From SDF1/CXCR4 Signaling Axis in Normal Hematopoietic Cells.

Abstract 2383

Poster Board II-360

Elevated expression of CXCR4, a receptor for SDF1, with Internal Tandem Duplication of Flt3 (ITD-Flt3) is an indicator of poor prognosis in patients with acute myeloid leukemia. We previously showed that ITD-Flt3 enhances migration of hematopoietic cells to SDF1, suggesting that ITD-Flt3 may facilitate dissemination of leukemia cells by modulating SDF1/CXCR4 signaling and that blocking this functional cross-talk between ITD-Flt3 and CXCR4 pathways may have therapeutic benefit. While identification of selective pathways in cells transformed by ITD-Flt3, which are distinct from normal cells is crucial to develop therapeutic agents without hematopoietic toxicity, the mechanisms responsible for aberrant migration induced by ITD-Flt3 are not known. We now demonstrate the existence of CXCR4 signaling pathways regulated by ITD-Flt3 that are distinct from normal CXCR4 signaling using genome wide transcription analysis.

Ectopic expression of ITD-Flt3 in Ba/F3 cells enhances random cell migration and modulates expression of 1,675 genes out of 41,174 genes (4.1%) examined compared to control cells lacking ITD-Flt3. ITD-Flt3 down-regulated CXCR4 mRNA by 55% compared to control and modulated 36 additional transcripts implicated in cell migration and localization (0.09%, P<0.05), further substantiating a role of ITD-Flt3 in cell migration and suggesting that enhanced migration was not dependent on CXCR4. Coordinately, ectopic ITD-Flt3 significantly enhanced cell migration to SDF1 compared to cells expressing wild-type Flt3 despite reduction in CXCR4. In contrast, in primary HPC, enhanced migration induced by ITD-Flt3 was inhibited by conditional deletion of CXCR4 in Cre-ER^TM-CXCR4^fl/fl mice cells or by incubation with the selective CXCR4 antagonist AMD3100.

Analysis of gene expression in control and ITD-Flt3 expressing Ba/F3 cells migrating to SDF1 indicated that SDF1 modulates 1,647 (4.0%) of 41,174 genes analyzed in cells expressing ITD-Flt3, of which 1,190 genes (2.5%) were regulated by SDF1 exclusively in ITD-Flt3 cells, such as neurofibromatosis type 1 and p27^kip1. SDF1 regulated 457 genes (1.1%) in both cells expressing wild-type Flt3 and ITD Flt3, whereas, 384 (0.5%) transcripts were regulated by SDF1 in wild-type Flt3 expressing cells but not in ITD-Flt3 cells. Genes associated with transcription, cytoskeleton, ubiquitination, kinase and iron transport were functionally clustered both in ITD-Flt3 and wild-type Flt3 cells migrating to SDF1. In contrast, genes involved in apoptosis, cell cycle, glycosylation and dephosphorylation were significantly enriched in ITD-Flt3 but not in wild-type Flt3 cells, suggesting that ITD-Flt3 generates CXCR4 signaling pathways that are qualitatively different from normal cells. In addition, 69 genes (0.17%) were down-regulated by SDF1 in ITD-Flt3 cells but up-regulated in control cells or vice versa, including Rho-associated coiled-coil forming kinase 1 (ROCK1), which has been linked with leukocyte transendothelial migration, cell motility and tumor cell invasion. While baseline expression of ROCK1 was not affected by the presence of ITD-Flt3, ROCK1 was up-regulated 251% by SDF1 in control cells but down-regulated by 67% in ITD-Flt3 cells. The ROCK antagonist Y27632 significantly decreased migration of control Ba/F3 cells lacking ITD-Flt3 to SDF1 (77 ± 6% inhibition, P<0.01), but had no effect on SDF1-induced migration of ITD-Flt3 cells, suggesting that an increase of ROCK1 is required for normal CXCR4 signaling, while enhanced migration to SDF1 induced by ITD-Flt3 is independent of ROCK system.

Our data provide additional evidence for functional cross-talk between SDF1/CXCR4 and ITD-Flt3 signaling pathways. Inhibition of aberrant migration to SDF1 induced by ITD-Flt3 by blocking CXCR4 suggests that antagonizing CXCR4 may inhibit dissemination of hematopoietic cells expressing ITD-Flt3. ITD-Flt3 regulates overlapping but functionally distinct pathways down-stream of SDF1/CXCR4 compared to those in cells without ITD-Flt3. Genes differentially regulated by SDF1 specifically in ITD-Flt3 cells may represent key targets regulating aberrant migration by ITD-Flt3 in response to SDF1 to prevent unnecessary dissemination and invasion of ITD-Flt3⁺ acute leukemia cells without affecting normal hematopoiesis.