FIP1L1/PDGFRα Imposes Commitment towards Eosinophil Lineage on Hematopoietic Stem/Progenitor Cells by Modifying the Expression and Function of Lineage Specific Transcription Factors.

Abstract

Constitutively activated tyrosine kinases (TKs) cause various types of leukemia. For example, BCR/ABL and TEL/PDGFRβ (T/PRβ) are the causative genes of CML and CMML, respectively. However, these leukemogenic TKs (LTKs) activate common sets of downstream molecules such as Ras/MAPK, PI3-K/Akt, and STATs. So, it remains unknown how disease phenotypes (i.e., the lineage and differentiation stage of leukemic cells and the clinical course of the patient) are determined by LTKs. One possibility is that LTK by itself has a potential to promote the development of leukemic cells specific for each LTK. Alternatively, these phenotypes might originate from the lineage and/or differentiation stage of leukemic stem cells, in which each LTK was initially generated. To characterize these mechanisms, in this study, we utilized FIP1L1/PDGFRα (F/PRα), a causative gene of hyper eosinophilic syndrome (HES)/chronic eosiinophilic leukemia (CEL). First, we transduced F/PRα and T/PRβ into murine c-KithighSca-1+Lin− (KSL) cells using the retrovirus system. As a result, both LTKs enabled KSLs to grow under factor-deprived conditions. Next, we examined the effects of these LTKs on the development of Gr1+IL-5 receptor(R)+ eosinophil progenitors (EoPs) from KSL cells. After 6-day cultures with SCF, TPO, IL-6, and FLT3L, 52% of F/PRα-transfected KSL cells became to be Gr1+IL-5R+, while only 4% and 15% of the cultured cells were Gr1+IL-5R+ in mock- and T/PRβ-transfected KSL cells, respectively. Similarly, F/PRα preferentially developed EoPs from common myeloid progenitor (CMP)s. Furthermore, when expressed in megakaryocyte/erythrocyte progenitor (MEP)s and common lymphoid progenitor (CLP)s, F/PRα inhibited the development of erythroid cells, megakaryocytes and B lymphocytes, respectively. Importantly, F/PRα but not T/PRβ aberrantly developed EoPs from MEPs and CLPs. However, F/PRα was not able to immortalize CMP, granulocyte/monocyte progenitor (GMP), MEP, or CLP. These results suggest that, although F/PRα can reprogram these progenitors towards Eo lineage, it must be generated in hematopoietic stem cells to cause HES/CEL. Next, we expressed chimeric LTKs (F/PRβ and T/PRα) and two activated forms of PRα each harboring point mutation detected in gastrointestinal stromal tumors (GIST) (PRαV561D and PRαD842V) in KSL cells. As a result, all these TKs enabled KSL cells to proliferate under factor-deprived conditions. However, only T/PRα promoted the development of EoP from KSL cells, indicating that specific activity derived from chimeric PRα is necessary to cause HES/CEL. Finally, we examined the effects of F/PRα on the expression and function of lineage specific transcription factors by RT-PCR analysis and luciferase assays. When expressed in KSL cells, F/PRα augmented the expression of C/EBPα and GATA-2 and decreased PU.1 expression in KSL cells as early as 24h prior to the apparent development of EoPs as compared with mock and T/PRβ. Furthermore, we found that F/PRα and its downstream Ras but not STAT5 or PI3-K inhibited the activity of PU.1, but not of GATA-2 or C/EBPα. Together, these results suggest that F/PRα selectively induces EoP from hematopoietic stem/progenitor cells by modifying the expression and function of lineage specific transcription factors.