Internal Tandem Duplication Mutations of Flt3 (ITD-Flt3) up-Regulate p21 WAF1/CDKN1 Protein That Negatively Regulates Aberrant Proliferation of Hematopoietic Cells Induced by ITD-Flt3.
Authors: Abe Mariko , Pelus Louis M. , Taketani Takeshi , Yamaguchi Seiji , Fukuda Seiji 
Published: October 2019

Abstract
Abstract 5037
Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) are frequently found in patients with acute myeloid leukemia (AML) and are associated with poor prognosis. However, lack of significant efficacy in patients with AML treated with ITD-Flt3 inhibitors in several clinical trials underscores the need for identification of pathways down-stream of ITD-Flt3 that are distinct from normal hematopoietic cells in order to develop novel therapeutic approaches. We have shown that ITD-Flt3 induces expression of the antiapoptotic protein Survivin and cyclin dependent kinase inhibitor (CDKI) p21WAF1/CDKN1 (p21) and that antagonizing Survivin in primary mouse hematopoietic progenitor cells (HPC) expressing ITD-Flt3 inhibits growth factor-independent proliferation (Fukuda et al. Blood 2009). Similarly, ectopic Survivin increases proliferation of mouse primary HPC and this enhancing effect is absent when p21 is functionally deleted (Fukuda et al. Blood 2004). These previous findings suggest that p21 lies down stream of Survivin and involved in facilitating growth factor independent HPC proliferation mediated by ITD-Flt3 signaling. However, analysis of p21 function in growth factor-independent ITD-Flt3 transduced HPC now indicates that p21 in fact negatively regulates ITD-Flt3 signaling.
Flt3 ligand (FL) induced marginal proliferation and p21 protein expression in Ba/F3 cells expressing wild-type Flt3. Expression of ITD-Flt3 significantly enhanced proliferation, cell cycle progression and inhibition of apoptosis coincident with up-regulation of p21 in Ba/F3 cells in the absence of FL or IL-3. In contrast, down-regulation of p27Kip1 by ITD-Flt3 was coincidently observed. In addition, higher levels of p21 were observed in ITD-Flt3+ MV4-11 human acute leukemia cells compared to ITD-Flt3- RS4;11 cells. Treatment of MV4-11 cells and Ba/F3 cells expressing ITD-Flt3 with the ITD-Flt3 inhibitor AG1296 significantly reduced p21 expression. In BaF3 cells, elevated p21 expression induced by ITD-Flt3 was suppressed by selective inhibitors for protein kinase A (H89), MAPK (PD98059), PI3-kinase (LY294002), Akt (Akt inhibitor) and p53 (pifithrin-α), suggesting involvement of these signaling pathways in ITD-Flt3 mediated p21 up-regulation.
Although ITD-Flt3 induces p21 expression and growth factor-independent proliferation in Ba/F3 cells, control cells expressing both ectopic wild-type Flt3 and p21 did not proliferate in the absence of growth factors, indicating that p21 is not sufficient to substitute for ITD-Flt3. In Ba/F3 cells expressing ITD-Flt3, p21 knock down by shRNA enhanced growth factor-independent proliferation. Ectopic expression of ITD-Flt3 in CFU-GM from p21+/+ mice resulted in significant growth of growth factor-independent CFU-GM in vitro, which was further accelerated by gene deletion of p21 (170 % increase: P<0.05), while co-expression of p21 with ITD-Flt3 in bone marrow cells from p21+/+ mice dramatically decreased growth factor-independent proliferation of c-kit+, Sca-1+, lin- cells (80±6% reduction: P<0.01). In contrast, Survivin gene deletion significantly reduced growth factor-independent CFU-GM proliferation by 74% (P<0.05).
Although ITD-Flt3 up-regulates p21 and Survivin independent of hematopoietic growth factors in primary HPC transformed by ITD-Flt3, p21 negatively regulates aberrant growth factor-independent proliferation. This is in contrast to Survivin, which functions up-stream of p21 in normal HPC proliferation and positively regulates ITD-Flt3 signaling. This suggests that p21 antagonizes Survivin down-stream of ITD-Flt3 signaling but cooperates/synergizes with Survivin to enhance proliferation in normal HPC. Since p21 deficiency has been shown to result in fewer total and S-phase CFU and ectopic expression of p21 in p21 −/− bone marrow cells restores total and cycling CFU, p21 positively regulates cell cycle and proliferation of normal HPC, but negatively regulates growth factor-independent proliferation of CFU induced by ITD-Flt3. Our data also suggests that CDKIs p21 and p27 have divergent function in ITD-Flt3 signaling. Manipulation of p21 signaling based on their functional difference between normal and transformed HPC may represent an alternative therapeutic strategy for hematological malignancies expressing ITD-Flt3 that are refractory to ITD-Flt3 inhibitors.

Disclosures
No relevant conflicts of interest to declare.