p21^WAF1/CDKN1 Function In Hematopoietic Progenitor Cell Proliferation Is Inversely Regulated by Wild-Type Flt3 and Internal Tandem Duplication Mutation of Flt3

Abstract 4175

Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) induce constitutive activation of Flt3 signaling and are frequently found in acute myeloid leukemia patients with poor prognosis. We have previously reported that Survivin enhances proliferation of mouse primary hematopoietic progenitor cells (HPC) and that this enhancing effect on HPC proliferation is absent when p21^WAF1/CDK1 (p21) is deleted, suggesting that p21 is required for Survivin to enhance normal HPC proliferation by hematopoietic growth factors (Fukuda et al. Blood 2004). Our subsequent studies showed that several ITD-Flt3 mutations increase expression of both Survivin and p21 and enhance growth factor independent HPC proliferation through up regulation of Survivin (Fukuda et al. Blood 2009). These findings suggest that p21 may also regulate growth factor independent HPC proliferation mediated by ITD-Flt3. However, our previous studies and those of others suggest that p21 function can vary and affect cell proliferation in diverse ways depending on cell type, and that intracellular signals generated by the wild-type Flt3 and ITD-Flt3 receptors can be qualitatively different. In the present study, we evaluated functional differences in the Survivin/p21 axis between wild-type Flt3 versus ITD-Flt3 signaling on HPC proliferation using p21^-/- mouse bone marrow cells.

P21 gene deletion significantly enhances growth factor independent proliferation of ITD-Flt3 transduced CFU-GM in vitro compared to p21^+/+ mice (221±5% increase: P<0.02), suggesting that p21 inhibits growth factor independent proliferation of HPC by ITD-Flt3. This is in contrast to Survivin deletion, which significantly reduced growth factor-independent CFU-GM proliferation (74% reduction, P<0.05). Similar to ITD-Flt3, Flt3 ligand (FL) induced marginal expression of p21 in Ba/F3 cells expressing wild-type Flt3 and stimulated proliferation of p21^+/+ CFU-GM transduced with wild-type Flt3. In contrast to the negative role of p21 in ITD-Flt3 mediated growth factor independent HPC proliferation, p21 deletion significantly reduced proliferation of CFU-GM over-expressing wild-type Flt3 and incubated with FL compared to p21^+/+ CFU-GM (59±6% reduction: P<0.01), indicating that p21 positively regulates proliferation of HPC stimulated by wild-type Flt3 signaling, which is consistent with our report and others where p21^-/- CFU-GM incubated with GM-CSF and/or SCF were significantly decreased compared to p21^+/+ cells (Mantel et al. Blood 1996, Fukuda et al. Blood 2004).

Our data indicates that while both wild-type Flt3 and ITD-Flt3 signaling up-regulate p21, wild-type Flt3 and ITD-Flt3 differentially modify p21 function that inversely regulates proliferation of HPC. The positive regulatory role of p21 in HPC proliferation by wild-type Flt3 signaling is in good agreement with our study that the Survivin/p21 axis positively regulates growth factor dependent HPC proliferation. This suggests that the Survivin/p21 axis may enhance HPC proliferation in the presence of wild-type Flt3 signaling. In contrast, p21 negatively regulates aberrant growth factor independent proliferation in primary HPC transformed by ITD-Flt3, whereas Survivin positively regulates ITD-Flt3 signaling, suggesting that the Survivin/p21 functional axis does not exist or play a role in aberrant growth factor independent proliferation by ITD-Flt3 signaling as opposed to normal HPC proliferation. Our study uncovers one functional difference between wild-type Flt3 and ITD-Flt3 signaling that may aid in developing specific therapeutic molecules for ITD-Flt3⁺ hematological malignancies that target ITD-Flt3⁺ transformed cells without affecting normal hematopoiesis.