Expansion of Normal and Leukemic Hematopoietic Progenitor Cells by PTH Requires bFGF Activation of c-Kit and Its Downstream JAK2/STAT5 Signaling.
Authors: Itkin Tomer , Ludin Aya , Schajnovitz Amir , Kalinkovich Alexander , Kollet Orit , Coffin Douglas J , Berg Thorsten , Piacibello Wanda , Lapidot Tsvee 
Published: October 2019

Abstract
Abstract 2511
Poster Board II-488

Introduction:
The signaling mechanisms which orchestrate hematopoietic stem and progenitor cell (HSPC) expansion, and proliferation of leukemic progenitor cells are not fully understood. In vivo parathyroid hormone (PTH) treatment was shown to expand HSPC via activation of bone forming osteoblasts (Calvi et al., Nature, 2003). PTH induced bone remodeling via activation of both osteoblasts and osteoclasts is a basic fibroblast growth factor (bFGF) dependent event (Okada et al., JBC, 2003; Hurley et al., BBRC, 2006). Different FGF receptors are expressed on various sub-types of HSPC and also on bone marrow (BM) stromal cells. This ligand, alone or in combination with other cytokines, promotes ex vivo expansion of both human and murine HSPC. Interestingly, bFGF mediated expansion of HSPC in vitro, in a culture of total BM cells, but not of purified Sca-1+/c-Kit+/Lineage− (SKL) murine HSPC (Yeoh et al., Stem Cells, 2006), implying that HSPC expansion by bFGF is also mediated indirectly via the microenvironment. KG1a is a human CD34+ progenitor AML cell line which harbors a mutation due to a chromosomal translocation, resulting in a constitutively active isoform of the FGF receptor (Gu et al., Blood, 2006). We used this cell line to elucidate the mechanism behind FGF mediated expansion of human CD34+ progenitors. The goal of this study is to reveal the roles of PTH and bFGF in regulation of normal and leukemic HSPC expansion in vivo and the downstream signaling machinery.

Results:
In order to understand how cytokines control HSPC proliferation and expansion in vivo, we repeatedly injected PTH to wild type (WT) and bFGF knock-out (KO) mice (5 injections per week) for 4 weeks. We observed that PTH administration to WT mice increased the levels of primitive SKL progenitors in the BM and spleen, decreased the percentage of G0 quiescent SKL cells, and upregulated c-Kit expression on the Lin−/Sca-1+ primitive progenitor population. Surprisingly, these effects were not observed in bFGF KO mice treated with PTH, revealing that PTH induced expansion of HSPC requires bFGF signaling. Repeated bFGF injections for only 1 week (total of 7 injections), significantly increased the numbers and frequency of hematopoietic progenitor cells (HPC) with only a minor effect on differentiated white blood cell numbers in the murine BM and spleen. HSPC expansion by bFGF was accompanied with splenomegaly, a phenotype resembling myelo-proliferative like diseases. In addition we found that bFGF downregulated SDF-1 and upregulated SCF expression on BM stromal cells together with upregulation of c-Kit expression on Lin−/Sca-1+ primitive progenitors. Inhibition of FGF signaling in KG1a cells using a specific FGFR inhibitor reduced STAT5 phosphorylation levels and increased the G0 quiescent cell fraction. Moreover, specific inhibitors of FGFR, JAK2 and STAT5, completely blocked human leukemic KG1a proliferation, implying for the involvement of this signaling pathway in expansion of immature human CD34+ AML cells.

Conclusions:
Our preliminary results reveal that bFGF is down stream of PTH induced HSPC expansion in vivo and that this ligand can expand progenitor cells in their natural BM and spleen microenvironments. bFGF rapidly mediates higher expansion of murine SKL HSPC within 1 week as compared to PTH expansion within 4 weeks. PTH and bFGF induced expansion is mediated both directly and indirectly by upregulating c-Kit receptors on HSPC and its ligand, SCF expression by stromal cells. Our in vitro experiments with malignant human KG1a cells indicate that FGF signaling activates STAT5 via JAK2 to promote expansion of both normal and leukemic progenitor cells. This activation is most probably mediated via SCF/c-Kit signaling since AML cells express high levels of these molecules (Ramenghi et al., Stem Cells, 1994) and therefore may bypass the dependency on the stromal microenvironment. Further studies will provide insights on how PTH/bFGF and their downstream mediators like c-Kit and SCF control normal and leukemic HSPC expansion and their evolving stromal microenvironments. These insights may be used for the development of drugs to inhibit expansion and survival of leukemic HSPC in patients. Moreover, they may also contribute to future development of therapeutic applications aimed at expanding normal HSPC prior to their mobilization or following clinical HSPC transplantation protocols.

Disclosures:
No relevant conflicts of interest to declare.