The Survivin/p21WAF1/Cip1 Axis and the CXC Chemokine SDF1α Regulate Growth Factor Independent Survival of Primary Hematopoietic Progenitor Cells Induced by Internal Tandem Duplication Mutations of Flt3.
Authors: Fukuda Seiji , Bian Huimin , Conway Edward M. , Pelus Louis M. 
Published: April 2020

Abstract
Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) and aberrant over-expression of the inhibitor of apoptosis protein Survivin, are frequently found in patients with acute myeloid leukemia (AML) and are associated with poor prognosis. We have shown that ITD-Flt3 increases expression of Survivin and the cyclin dependent kinase inhibitor p21WAF1/Cip1 in Ba/F3 cells and that Survivin regulates hematopoietic progenitor cell (HPC) proliferation through p21WAF1/Cip1 dependent and independent pathways (Fukuda et al. Blood, 2004.103:120), which functionally link Survivin and p21 downstream of ITD-Flt3 signaling. Elevated CXCR4 expression associated with ITD-Flt3 mutations has been recently reported in AML patients and may confer an adverse prognosis. Although it is not known whether ITD-Flt3 signaling interacts with the CXCR4 signaling pathway to regulate cell survival, our recent data indicate that ITD-Flt3 and SDF1α/CXCR4 signaling pathways functionally interact to enhance hematopoietic cell migration (Fukuda et al. Blood, 2005.105:3117). This suggests that these two pathways may coordinate or regulate trafficking of transformed hematopoietic cells and may be involved in AML disease progression. In order to investigate whether Survivin, p21 and SDF1α/CXCR4 mediate enhanced HPC survival as a consequence of constitutive ITD-Flt3 signaling, we utilized marrow cells from control, p21−/− or Tamoxifen-Cre conditional Survivinfloxed/floxed knockout mice and evaluated if disrupting Survivin or p21 can block the aberrant enhanced HPC proliferation induced by ITD-Flt3 in vitro and whether SDF1α could accentuate the enhanced survival of primary mouse HPC induced by ITD-Flt3. Over-expression of human wild-type Flt3 failed to induce growth factor independent proliferation of marrow CFU-GM, however, ectopic expression of ITD-Flt3 mutants cloned from AML patients significantly enhanced survival of CFU-GM from control mice after growth factor deprivation and allowed for factor independent proliferation. In contrast, conditional deletion of Survivin or loss of p21 significantly reduced growth factor independent survival of CFU-GM induced by ITD-Flt3 (53±17% and 41±9% reduction in Survivin and p21 KO cells, respectively; P&lt;0.05), suggesting that Survivin and p21 are required for enhanced survival of HPC induced by ITD-Flt3. Furthermore, SDF-1α dose-dependently increased the number of growth factor independent CFU-GM expressing ITD-Flt3 (40–140% increase), suggesting that SDF-1α/CXCR4 and ITD-Flt3 signaling functionally interact to enhance HPC survival, an effect consistent with the synergistic enhancement of normal HPC survival induced by the combination of Flt3 ligand plus SDF-1α and enhanced migration to SDF1α induced by ITD-Flt3. This effect is unlikely due to up-regulation of CXCR4, since CXCR4 in Ba/F3 and 32D cells expressing ITD-Flt3 was significantly down-regulated compared to wild type Flt3. These reults provide evidence that the Survivin/p21 axis and SDF1α/CXCR4 mediate resistance to apoptosis and transformation of HPC and/or cell trafficking by ITD-Flt3, and that disruption of these signaling pathways may be additional therapeutic strategies for patients with AML expressing ITD-Flt3.