FLT3-ITD Knock-in Impairs Hematopoietic Stem Cell Quiescence/Homeostasis, Leading to a Myeloproliferative Neoplasm
Authors: Chu S. Haihua , Heiser Diane , Li Li , Kaplan Ian M , Huso David , Civin Curt I. , Small Donald 
Published: October 2019

Abstract 46

In murine hematopoiesis, long-term hematopoietic stem cells (LT-HSCs) are classically defined as Fms-like tyrosine kinase (Flt3) negative with Flt3 positivity often serving as a marker for differentiation and loss of self- renewal capacity. Flt3 has previously been shown to be dispensable for HSC maintenance and myeloid development. Flt3 and Flt3-ligand knockout mice have minor defects in overall hematopoiesis. However, these studies fail to take into account the effects of overlapping or compensatory pathways that may mask important roles Flt3 may play in HSC homeostasis. To date, few studies have addressed the effects of constitutive Flt3 activity on normal hematopoiesis or stem cell function. These mutations in FLT3 are of particular importance as they comprise one of the most common molecular alterations found in human acute myeloid leukemias (AML). Internal tandem duplication (ITD) mutations in FLT3 render the receptor tyrosine kinase constitutively active and portend a poor prognosis for those patients carrying the mutation. Many of these patients respond only transiently to treatment, possibly due to a reservoir of stem-like cells that are able to escape therapy. To study the effects of expression of constitutive activation mutations of FLT3 in primitive HSC compartments, our lab has developed a murine knock-in model in which this mutation is expressed under control of the endogenous murine Flt3 promoter. Flt3-ITD mice develop a myeloproliferative neoplasm (MPN), display imbalances in hematopoietic stem and progenitor cell population distributions (of note, 2–5 fold reductions in primitive LT-HSCs in SLAM (LIN-CD150+CD48−) and KSL-SLAM (Kit+Sca+Lin-CD150+CD48−) populations) and HSC defects (as measured by significantly reduced engraftment potential). Here, we show these perturbations to be the result of a Flt3-ITD-driven, cell-autonomous, overproliferation within a compartment of normally quiescent LT-HSCs, as evidenced by increased BrdU incorporation, cell cycle entry and upregulation of genes that promote cell cycle (Pim1, Ccnd1). This rapid HSC exhaustion and depletion of the LT-HSC compartment is coupled to the activation of downstream Flt3 signaling, most prominently through Stat5 with observed increases in expression of Stat5 targets (e.g. Fos, JunB, Mkp1, Socs3). Furthermore, for the first time, we show that Flt3 is expressed at detectable (albeit low) levels in this MPN-initiating, LT-HSC cell population and capable of exerting significant functional effects in this compartment. Surprisingly, the depletion and defects in HSC function are completely reversible upon treatment with the small molecule inhibitor, sorafenib, while simultaneously leading to near complete ablation of disease. Taken together, our data implicate previously unrecognized roles for Flt3 in the extremely dynamic process of stem cell homeostasis and quiescence, which appears closely coupled to pathogenesis, as well as pointing to Flt3 as an attractive target for targeted therapy.

Disclosures:

No relevant conflicts of interest to declare.