KIT Receptor Gain-of-Function in Hematopoiesis Enhances Stem Cell Self-Renewal and Promotes Progenitor Cell Expansion

Authors


  • Author contributions: S.D.: conception and design, collection of data and analysis and interpretation, and manuscript writing; B.B.: conception and design and manuscript writing; Y.Y.: collection and assembly of data; C.Y.P.: design, data analysis, and interpretation; M.A.S.M.: design, data analysis, and interpretation and manuscript writing; P.B.: conception and design, data analysis and interpretation, manuscript writing, financial support, and final approval of manuscript.

Abstract

The KIT receptor tyrosine kinase has important roles in hematopoiesis. We have recently produced a mouse model for imatinib resistant gastrointestinal stromal tumor (GIST) carrying the KitV558Δ and KitT669I (human KITT670I) mutations found in imatinib-resistant GIST. The KitV558Δ;T669I/+ mice developed microcytic erythrocytosis with an increase in erythroid progenitor numbers, a phenotype previously seen only in mouse models of polycythemia vera with alterations in Epo or Jak2. Significantly, the increased hematocrit observed in KitV558Δ;T669I/+ mice normalized upon splenectomy. In accordance with increased erythroid progenitors, myeloerythroid progenitor numbers were also elevated in the KitV558Δ;T669I/+ mice. Hematopoietic stem cell (HSC) numbers in the bone marrow (BM) of KitV558Δ;T669I/+ mice were unchanged in comparison to wild-type mice. However, increased HSC numbers were observed in fetal livers and the spleen and peripheral blood of adult KitV558Δ;T669I/+ mice. Importantly, HSC from KitV558Δ;T669I/+ BM had a competitive advantage over wild-type HSC. In response to 5-fluorouracil treatment, elevated numbers of dividing LinSca+ cells were found in the KitV558Δ;T669I/+ BM compared to wild type. Our study demonstrates that signaling from the KitV558Δ;T669I/+ receptor has important consequences in hematopoiesis enhancing HSC self-renewal and resulting in increased erythropoiesis. STEM Cells 2013;31:1683- -1695

Introduction

The hematopoietic stem cell (HSC) compartment in the bone marrow (BM) maintains steady state hematopoietic numbers by self-renewal and differentiation into progenitors. HSCs, based on their self-renewing abilities, have been distinguished into long-term HSCs (LT-HSC), short-term HSCs (ST-HSC), and finally multipotent progenitor (MPP) cells [1]. The receptor tyrosine kinase KIT and its cognate ligand KITL have long been known to be critical components of the BM niche. LT-HSC and ST-HSC from Kit loss-of-function mutations such as Wv, W41, and W42 in either homozygous or heterozygous conditions are ineffective at reconstituting BM in irradiated recipients indicating an important role for KIT in stem cell self-renewal and survival [2].

Apart from its role in stem cell function, the KIT-KITL pair is also known to have a role in regulating adult definitive erythropoiesis. The mutations in the Kit (W) and KitL (Sl) genes have provided important insights into the role of KIT in erythropoiesis. Mice with partial loss-of-function mutations in either gene are viable but have macrocytic anemia while more severe mutations lead to embryonic lethality due to lack of erythropoiesis [3–5]. KIT signaling is especially important in the proliferation and survival of erythroid progenitors, specifically at the burst-forming-unit-erythroid (BFU-E) stage [6, 7].

While much is known about hematopoietic phenotypes of Kit deficient mutant mice, relatively little information is known about the consequences of Kit gain-of-function mutations [8]. Somatic activation loop Kit mutations such as KitD816V are observed in human mastocytosis, AML, and seminoma. BAC transgenic mice carrying the KitD816V mutation die perinatally as a result of strong hematopoietic phenotypes [9]. In contrast, human patients with somatic or familial gastrointestinal stromal tumor (GIST) most often carry juxtamembrane domain mutations and as in KitV558Δ/+ mice only weak hematopoietic phenotypes are observed [10]. Recently, we engineered mice bearing in addition to the KitV558Δ mutation a “gatekeeper” KitT669I second site mutation (human T670I), KitV558Δ;T669I/+, as observed in imatinib-resistant human GIST [11]. The KitV558Δ;T669I/+ mice developed small cecal GIST lesions with pronounced interstitial cell of Cajal hyperplasia in the stomach and colon. Interestingly, these mice showed increased hematocrit (HCT) and BFU-E numbers with features reminiscent of mice with polycythemia vera (PV) and other myeloproliferative neoplasms [11].

In this study, we have investigated the consequences of the KitV558Δ;T669I gain-of-function mutation on hematopoiesis in mutant mice. We have assessed erythroid progenitor numbers by flow cytometry, analyzed whether splenectomy could normalize HCT numbers in KitV558Δ;T669I/+ mice, and then investigated whether the mutation resulted in the expansion of myeloid progenitors and stem cells. We also investigated if stem cell function is altered in the KitV558Δ;T669I/+ mice by transplantation assays and analysis of stem cell proliferation. Our data show that the KitV558Δ;T669I mutation results in expansion of erythroid and myeloid progenitors and in particular enhances HSC self-renewal.

Materials and Methods

Experimental Animals

The KitV558Δ;T669I/+ mice on the C57BL/6J background have been described previously and genotyped according to the published protocol [11]. Congenic B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Peripheral blood was collected from the retro-orbital cavity or the tail vein using EDTA-treated glass capillaries and blood parameters were analyzed using Hemavet 950 (Drew Scientific, Waterbury, TX, http://www.drew-scientific.com). All animal procedures were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center.

Progenitor Cell Analysis

Colony-forming assay: unfractionated BM (50,000/plate) or spleen (100,000/plate) cells from at least three mice were plated in 1 ml Iscove's modified Dulbecco's medium medium containing 1.2% (wt/vol) methylcellulose, 30% fetal calf serum, 2 mmoL/l glutamine, 0.1 mmoL/l 2-mercaptoethanol, and 4 mmoL/l hemin with cytokines (erythropoietin (EPO) 6 U/ml, IL3 100 ng/ml, and KitL 100 ng/ml). Cultures were maintained in triplicate at 37°C in humidified 5% CO2. Granulocyte-monocyte (GM) and granulocyte-erythrocyte-monocyte-megakaryocyte (GEMM) colonies were scored after 7–10 days of culture. Colonies were scored using Nikon Eclipse TE 200 and images were taken using Nikon Eclipse Ti-S.

CFU-S: C57BL/6 mice used as recipients were irradiated (10 Gy: 5 Gy and 5 Gy, 3-hour apart) on the day of transplantation and injected with 100,000 wild-type or KitV558Δ;T669I/+ BM cells. Mice were euthanized at the end of 11 days or when they appeared very morbid and spleens were immersed in Bouin's solution for analysis.

Flow Cytometry

Single-cell suspensions from BM or spleen were prepared by crushing the tissues in a mortar and pestle in phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) and straining the cell mix through a 40 μm cell strainer. Nucleated cells were counted in methylene blue with 1% acetic acid (Life Technologies, Grand Island, NY, http://www.lifetechnologies.com) using a hemacytometer. Peripheral blood was collected by retro-orbital or tail vein bleeding and subjected to osmotic lysis using ammonium chloride buffer (Stemcell Technologies, Vancouver, Canada, http://www.stemcell.com). Wild-type and KitV558Δ;T669I/+ embryos were obtained by crossing KitV558Δ;T669I/+ males with C57BL/6 females. Fetal livers (FL) were excised from the embryos at dpc 14.5. A single-cell suspension of FL cells was obtained by gently mashing the FL on a 40 μm cell strainer in PBS containing 2% FBS. BM, spleen, and FL cells were stained in PBS buffer containing 2% FBS and the complete list of antibodies used is provided in Supporting Information. 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) or 7AAD (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com) was used to electronically gate live cells. Erythroid progenitors were analyzed by staining with Ter119 and CD71 antibodies as described previously [12]. HSC in BM, spleen, and peripheral blood were stained with the following lineage antibodies: CD3, CD4, CD8, B220, Ter119, Gr1, and Mac1. LT-HSC were defined as LinKit+Sca+CD34CD150+. FL-HSC were stained using a similar lineage cocktail with the omission of Mac1. FL-HSC were gated as LinKit+Sca+CD48CD150+. Myeloid progenitors were stained using the lineage cocktail of LT-HSC in addition to IL7Ralpha, IgM, and CD19. The progenitors were gated as megakaryocyte/erythrocyte progenitors (MEP): LinKit+ScaCD34FcγR, common myeloid progenitors (CMP): LinKit+ScaCD34lowFcγRlow, and granulocyte/macrophage progenitors (GMP): LinKit+ScaCD34highFcγRhigh. CD45 isotype analysis was done using CD45.1 PE/APC/APCCy7 and CD45.2 FITC/APC/PerCP5.5 antibodies. Cells were analyzed using FACSCalibur (BD Biosciences) or CyAn ADP (Beckman Coulter, Brea, CA, https://www.beckmancoulter.com) and data were analyzed using FlowJo software (TreeStar, Ashland, OR, http://www.treestar.com).

Cell Cycle Analysis

Cell cycle analysis of stem cells and erythroid progenitors was performed using Ki67-FITC (BD Biosciences) and DAPI according to the manufacturer's instructions. Briefly, cells were stained with biotinylated lineage antibodies as described for LT-HSC staining in addition to Kit (APC), Sca (PECy7), and CD34 (PE). Cells were fixed and processed for Ki67-FITC and DAPI staining according to manufacturer's instructions. Gating for Ki67 was established by staining cells with lineage, Kit, Sca, and CD34 antibodies in addition to the isotype control antibody provided by the manufacturer. Cell cycle entry of LinKit+Sca+ and LinKit+Sca+CD34 cells was also determined by 5-bromo-2-deoxyuridine (BrdU) incorporation in conjunction with DAPI. Mice were injected intraperitoneally twice with BrdU 0.5 mg/6 g b.wt., at 6-hour interval and euthanized 18-hour after the last injection. BrdU incorporation was detected using APC-anti-BrdU kit as per the manufacturer's instructions (BD Biosciences). Briefly, cells were stained with lineage antibodies-biotin (Streptavidin-APCCy7), Kit (PE), Sca (PECy7), and CD34 (FITC), followed by fixation and staining with APC-anti-BrdU and DAPI. Cells were analyzed using CyAn ADP (Beckman Coulter). Cells positive for BrdU were gated as described earlier [13, 14].

Transplantation

Competitive repopulation assay was performed for in vivo analysis of stem cell function. Wild-type or KitV558Δ;T669I/+ BM cells were mixed in 1:1 ratio with age (8–10 weeks) and sex matched CD45.1 BM cells. CD45.1 female recipient mice were irradiated (10 Gy: 5 Gy and 5 Gy, 3-hour apart) on the day of transplantation and injected with one million BM cell mixture. In instances of secondary transplantation, primary transplanted mice were euthanized after 16 weeks, BM cells were pooled from at least three mice and indicated numbers of cells (two million or five million) were injected into irradiated CD45.1 recipients. To investigate whether the spleen had functional HSC, two million whole spleen cells from wild-type or KitV558Δ;T669I/+ mice were mixed with 200,000 CD45.1 BM competitor cells and transplanted into CD45.1 recipient mice. Reconstitution of donor (CD45.2) myeloid and lymphoid cells was analyzed by flow cytometry in the peripheral blood and wherever indicated in the BM and spleens of transplanted mice at 16 weeks post-transplantation. All transplanted mice were kept on Sulfatrim diet for at least 4 weeks. Percent chimerism was defined as (% CD45.2 donor cells) (100)/(% CD45.2 donor + % CD45.1 competitor cells) [15, 16].

Splenectomy

Wild-type and KitV558Δ;T669I/+ mice were anesthetized, the splenic vessel was tied up, and the spleen was surgically excised. Animals received buprenorphine for management of pain following surgery. Peripheral blood parameters were analyzed 2 weeks prior to the surgery to obtain presplenectomy values. After splenectomy, mice were allowed to recover for 2 weeks and peripheral blood parameters were analyzed every week for up to 8 weeks. At the end of 8 weeks, mice were euthanized and the BM was analyzed for erythroid progenitors by flow cytometry.

5-Fluorouracil Treatment

5-Fluorouracil (5-FU, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) 150 mg/kg b.wt. was administered to mice once intraperitoneally. Peripheral blood was drawn at regular intervals by retro-orbital bleeding to measure leukocyte number and HCT using Hemavet 950 (Drew Scientific). Mice were injected with BrdU as described above and cells harvested from BM were used for cell cycle analysis by flow cytometry using APC-anti-BrdU (BD Biosciences) and DAPI.

Statistical Analysis

Comparison between two groups was done by unpaired Students t test analysis using GraphPad Prism (version 5.0). Statistical significance was achieved when p < .05 indicated as following in the text *, p < .05; **, p < .01; ***, p < .001; p = N.S. (not significant).

Results

Increased Erythroid Generation in KitV558Δ;T669I/+ Mice and Effect of Splenectomy

Previously, we showed that the KitV558Δ;T669I/+ mice have cecal GIST lesions, gastric and colonic ICC hyperplasia, and have increased HCT values. In addition, the spleen cellularity is increased by 1.5-fold in mutant mice. In accordance with the increased HCT, BFU-E progenitors in the BM and the spleen were also elevated in the KitV558Δ;T669I/+ mice. BFU-E in KitV558Δ;T669I/+ mice similar to their wild-type counterparts were EPO-dependent but their growth was more sensitive to KITL than wild-type progenitors [11]. In order to gain insight into the various stages of erythroid maturation in wild-type and mutant mice, flow cytometric analysis of erythroid progenitors was performed as described previously [12]. Different stages of erythroid maturation were classified on the basis of Ter119 and CD71 expression in conjunction with size of the cells (defined by forward scatter [FSC] property) (Fig. 1C). Proerythroblasts or R1 progenitors are defined as Ter119loCD71hi. As the erythroblasts mature discrete populations can be identified based upon gradual decline in CD71 expression and FSC as A:Ter119hiCD71hiFSChi, B:Ter119hi, CD71medFSClo, and C:Ter119hiCD71loFSClo. Our results show that R1 progenitor frequency was higher in the mutant BM and significantly increased in the mutant spleen compared to wild-type (Fig. 1A). Total R1 progenitor numbers were also significantly increased in the KitV558Δ;T669I/+ BM and spleen (Fig. 1B). The total numbers of the erythroblast subsets A and B were also significantly increased in the mutant spleen compared to wild-type (Fig. 1B). In accordance with the increased R1 progenitors, spleen cytospins of KitV558Δ;T669I/+ mice showed increased number of erythrocytes (data not shown). To assess whether increase in R1 progenitor numbers was due to increased proliferation, cell cycle status of these cells was analyzed by Ki67 and DAPI staining followed by flow cytometry. In accordance with increased erythroid numbers in the KitV558Δ;T669I/+ spleen, we found significantly increased frequency of R1 progenitors in S/G2/M phase of the cell cycle in the KitV558Δ;T669I/+ spleen (Fig. 1D). The cell cycle status in the mutant BM however was not significantly different compared to wild-type (Fig. 1D). Given the increased number of erythroid progenitors in the spleen we sought to ascertain the role of the spleen in the development of increased HCT in the KitV558Δ;T669I/+ mice, by performing splenectomy on 10-week-old wild-type and KitV558Δ;T669I/+ mutant mice. Postsurgery, HCT values dropped in both the wild-type (55.92 ± 2.1 to 43.24 ± 2.38) and KitV558Δ;T669I/+ (84.48 ± 2.32 to 52.88 ± 3.28, p < .0001) mice, although the reduction was significantly greater in the KitV558Δ;T669I/+ mice. Interestingly, the reduced postsurgery HCT values of KitV558Δ;T669I/+ mice were maintained until the time point of last measurement, namely 8 weeks (Fig. 1E). This is in contrast to the JAK2V617F-GFP BM transplantation model of PV, where splenectomy caused a gradual decrease in HCT levels and only by 7 weeks normal wild-type HCT values were obtained [17]. Erythroid progenitors assessed in the BM of splenectomized wild-type and KitV558Δ;T669I/+ mice had similar numbers of R1 progenitors at 8 weeks postsplenectomy (Fig. 1F). The splenectomy results collectively suggest that the spleen contributes significantly toward elevating erythroid cell numbers in the KitV558Δ;T669I/+ mice. Together our results show significantly elevated erythropoiesis in the KitV558Δ;T669I/+ mice which is normalized by splenectomy. This may suggest a critical role for a combination of cell-intrinsic property and the spleen microenvironment in the expansion of the erythroid compartment in the KitV558Δ;T669I/+ mice.

Figure 1.

KitV558Δ;T669I/+ mice develop erythrocytosis which is remedied by splenectomy. Total BM and SP cells were prepared for staining with Ter119 and CD71 antibodies for flow cytometry. (A, B): Graphical representation of flow cytometry results shows increased frequency and total Ter119+CD71+ cells, especially R1 erythroid progenitor population in the KitV558Δ;T669I/+ (GTK) BM and spleen. Frequency and total numbers of A and B populations are also significantly increased in the mutant spleen compared to WT (n = 4–9). (C): Representative flow cytometric histograms of BM and spleen cells show cells labeled with Ter119 and CD71 antibodies. The frequency of R1 and Ter119hi populations in total BM or spleen cells is depicted. Ter119hi population was further analyzed into A, B, and C subsets and numbers indicate frequency of cells within total BM or spleen cells. (D): BM and spleen cells were stained with cell surface markers Ter119 and CD71 and processed for Ki67-DAPI staining. Erythroid R1 progenitor cycling measured by Ki67 flow cytometry in the BM and spleen revealed increased cycling of progenitors in the KitV558Δ;T669I/+ spleen compared to wild-type but no change was observed in the BM of the two groups (n = 3–5). (E): In order to assess the contribution of the spleen toward increased erythropoiesis in the KitV558Δ;T669I/+, wild-type and KitV558Δ;T669I/+ mice were splenectomized. HCT levels 2 weeks postsurgery were significantly reduced in KitV558Δ;T669I/+ splenectomized mice compared to presurgical values, while wild-type hematocrit levels did not decrease significantly (n = 5). (F): Flow cytometric analysis of BM from wild-type and KitV558Δ;T669I/+ by Ter119 and CD71 staining at 8 weeks postsplenectomy showed similar numbers of erythroid R1 progenitors in the BM of KitV558Δ;T669I/+ compared to wild-type splenectomized mice (n = 3). Data are representative of at least two independent experiments. n—indicates number of mice per genotype. Values are mean ± SEM. Abbreviations: BM, bone marrow; FSC, forward scatter; GTK, Kit(V558Δ;T669I/+); HCT, hematocrit; SP, spleen; WT, wild type.

Myeloid Progenitor Expansion in the KitV558Δ;T669I/+ Mice

The increased number of erythroid progenitors in the KitV558Δ;T669I/+ mice led us to investigate whether myeloid subsets were also increased in these mice. Flow cytometric analysis of granulocyte and monocyte-macrophage populations in the BM and spleen revealed increased frequency and total cell numbers of both these cell types in the KitV558Δ;T669I/+ mice (Fig. 2A--2C). In order to assess whether homeostasis was also altered upstream in the myeloid hierarchy, a detailed flow cytometric analysis of myeloid progenitors namely CMP, GMP, and MEP was carried out as described previously [18]. In the BM of wild-type and KitV558Δ;T669I/+ mice, while the frequency and total numbers of CMP and GMP populations did not differ significantly, the MEP population in the KitV558Δ;T669I/+ BM was significantly increased compared to wild-type in terms of both frequency and total numbers (Fig. 2D, 2E, 2H). In the KitV558Δ;T669I/+ spleen, the frequency of all three progenitor types was significantly increased compared to wild-type (Fig. 2F, 2H). A significant elevation in total CMP and MEP numbers was also observed in the KitV558Δ;T669I/+ spleen (Fig. 2G). In order to physiologically assess the expansion of some of these myeloid populations, in vitro colony assays were performed. GM colonies were increased twofold in the KitV558Δ;T669I/+ BM and several fold increased in the KitV558Δ;T669I/+ spleen. In addition, GEMM colonies were 12-fold greater in the KitV558Δ;T669I/+ spleen compared to wild-type reiterating the elevated expansion of myeloerythroid progenitors in the spleens of these mice (Supporting Information Table S1). Reconstitution assays such as CFU-S also provide a measure of in vivo progenitor function. It has been described previously that CFU-S day 8 correspond to MEPs, CFU-S day 9 to CMP, and CFU-S day 12 to a mixture of MPP and CMP [1, 2, 19]. Transplantation of BM cells from wild-type or KitV558Δ;T669I/+ mice into irradiated C57BL/6 mice showed no difference in day 11 CFU-S colonies between wild-type and KitV558Δ;T669I/+ (wild-type: 8.25 ± 4.11; KitV558Δ;T669I/+: 10 ± 3.36; p = N.S., n = 5) which is in accordance with unchanged CMP numbers in the BM of wild-type and mutant as assessed by flow cytometry (compare Fig. 2D, 2E). Investigation of the lymphoid lineages by flow cytometry interestingly revealed reduced frequency of B220+, CD4+, and CD8+ cells in the KitV558Δ;T669I/+ BM and spleen compared to wild-type (Fig. 3A, 3D); however, respective total numbers were not statistically different (Fig. 3B, 3E). In summary, our results show that the Kit gain-of-function mutation affects the myeloid lineage and results in significant expansion of myeloerythroid progenitors in the KitV558Δ;T669I/+ mice.

Figure 2.

Expansion of the myeloid lineage in the adult BM and spleen of KitV558Δ;T669I/+ mice. Cells from the BM and spleen were stained with Gr1 and Mac1 antibodies and analyzed by flow cytometry. (A): Analysis of Mac1+-Gr1+ population in the BM and spleen showed significantly increased frequency of granulocyte and monocyte populations in KitV558Δ;T669I/+ mice compared to wild-type (n = 4–6). (B): Total cell numbers of granulocyte and monocyte cells are significantly increased in the KitV558Δ;T669I/+ spleen compared to wild-type (n = 4–6). (C): Representative staining profiles show frequency of granulocytes and monocytes in total BM and spleen. (D, E): BM cells were stained against lineage markers and LinKit+Sca cells were distinguished into CMP, GMP, and MEP subsets according to their CD34 and FcγRII/III surface expression. Flow cytometric analysis showed increased frequency of MEP in the mutant BM compared to wild-type. Total MEP numbers in the mutant BM were increased fivefold over wild-type (n = 3–4). (F, G): Spleen cells from wild-type and mutant stained for myeloid progenitor markers showed increased frequency of CMP, GMP, and MEP subsets in the KitV558Δ;T669I/+ spleen. While total CMP numbers were significantly increased in the mutant spleen, MEP numbers were remarkably (20-fold) higher in KitV558Δ;T669I/+ compared to wild-type (n = 3–4). (H): Representative flow cytometry plot for myeloerythroid progenitors. LinScaKit+ cells were divided into GMP, CMP, and MEP subsets. Numbers indicate frequency of cells in total BM and spleen. Data are representative of at least two independent experiments. Values are mean ± SEM. n—indicates number of mice. Abbreviations: BM, bone marrow; CMP, common myeloid progenitors; GMP, granulocyte/macrophage progenitors; GTK, Kit(V558Δ;T669I/+); MEP, megakaryocyte/erythrocyte progenitors; SP, spleen; WT, wild type.

KitV558Δ;T669I/+ Mutation Results in Increased Stem Cell Numbers at Extramedullary Sites and in the FL

We first sought to determine whether in addition to the expansion of MEPs in the KitV558Δ;T669I/+ mice, there was a change in the HSC-enriched LinSca+Kit+ (LSK) population. Interestingly, flow cytometric analysis revealed no significant differences in LSK frequency or numbers in the wild-type and KitV558Δ;T669I/+ BM (Fig. 4A, 4B). LSK frequency and total LSK numbers however were significantly higher in the KitV558Δ;T669I/+ spleen compared to wild-type (Fig. 4A, 4B). Based on these results, we further analyzed if LT-HSC compartment defined phenotypically as LinSca+Kit+CD34CD150+ [20] was altered. While no significant differences in the LT-HSC numbers of wild-type and KitV558Δ;T669I/+ BM (Fig. 4C, 4D) were observed, as with the LSK subset interestingly LT-HSC frequency and total cell numbers were significantly higher in the KitV558Δ;T669I/+ spleen compared to wild-type (Fig. 4C, 4D). In addition higher frequency of LT-HSC was also found in the peripheral blood of KitV558Δ;T669I/+ mice (Fig. 4E). These results show elevated HSC numbers in KitV558Δ;T669I/+ mice with dissemination of stem cells from their primary location, that is, the BM, to extramedullary sites. Previous studies have defined a clear role for KIT and KITL in the expansion of FL-HSC [21, 22]. At 14.5 dpc, KitV558Δ;T669I/+ FLs had significantly greater cellularity compared to wild-type (Fig. 5A). At this fetal age, equivalent numbers of LSK progenitor cells were observed in FLs of KitV558Δ;T669I/+ and wild-type embryos (Fig. 5B, 5C). We next investigated LT-HSC in the FL using the markers LinSca+Kit+CD150+CD48 [23, 24]. Interestingly, similar to the KitV558Δ;T669I/+ spleen, LT-HSC frequency and total cell numbers were significantly higher in the KitV558Δ;T669I/+ FLs (Fig. 5D, 5E). Lineage analysis also revealed significantly higher number of R1 erythroid progenitors in KitV558Δ;T669I/+ FLs (Fig. 5F). However, total B220+ or Gr1+ cells were similar in KitV558Δ;T669I/+ and wild-type FLs (Fig. 5G, 5H).

Figure 3.

Lymphopoiesis in KitV558Δ;T669I/+ mice. (A, B): BM and spleen cells were stained with B220 antibody and analyzed by flow cytometry. The frequency of B220+ cells was significantly reduced in the KitV558Δ;T669I/+ BM and spleen compared to WT (n = 3–5); however, total cell numbers were not significantly reduced compared to WT (n = 3–5). (C): Representative staining profile shows frequency of B220+ cells in BM and spleen. (D, E): The frequency of CD4+ and CD8+ cells was reduced in the KitV558Δ;T669I/+ BM and spleen; however, total cell numbers were not significantly reduced compared to WT (n = 3–5). (F): Representative staining profile shows frequency of CD4+ and CD8+ subsets in BM and spleen. Data are representative of at least two independent experiments. Values are mean ± SEM. n—indicates number of mice. Abbreviations: BM, bone marrow; FSC, forward scatter; GTK, Kit(V558Δ;T669I/+); SP, spleen; WT, wild type.

Figure 4.

Analysis of the stem cell compartment in wild-type and KitV558Δ;T669I/+ mice. (A, B): Total BM and spleen cells were stained against lineage cocktail and gated as LSK. LSK frequency and total cell numbers were not significantly altered in the KitV558Δ;T669I/+ BM compared to wild-type BM but in the KitV558Δ;T669I/+ spleen total LSK numbers were 11-fold elevated compared to wild-type spleen (n = 5). (C, D): LT-HSC defined as LinSca+Kit+CD150+CD34 were significantly higher in frequency and total cell numbers were sixfold higher in KitV558Δ;T669I/+ spleen compared to wild-type spleen. LT-HSC numbers were not significantly different in the BM of the two mice (n = 4–5). (E): The frequency of LT-HSC numbers was increased in the peripheral blood of KitV558Δ;T669I/+ compared to wild-type mice (n = 4). (F): Representative staining profiles for LSK and LT-HSC from BM and spleen. Lineage cells were electronically gated into LSK subset. LSK cells were subdivided into LT-HSC, MPP, and LMPP. Numbers represent frequency of cells in total BM or spleen. Data are representative of at least three independent experiments. Values are mean ± SEM. n—indicates number of mice. Abbreviations: BM, bone marrow; GTK, Kit(V558Δ;T669I/+); LT-HSC, long-term hematopoietic stem cell; LMPP, lymphoid-primed multipotent progenitors; LSK, LinSca+Kit+; MPP, multipotent progenitors; SP, spleen; WT, wild type.

Increased Self-Renewal of HSCs in the KitV558Δ;T669I/+ Mice

Whereas relatively mild Kit loss-of-function mutations had been shown to diminish HSC function, it was unclear how Kit gain-of-function mutations would affect HSC function [2]. To investigate whether the KitV558Δ;T669I mutation affected stem cell function, we carried out competitive repopulation assays. In primary transplantation experiments, 5 × 105 BM cells from wild-type or KitV558Δ;T669I/+ were competed with an equal number of congenic CD45.1 BM cells. At 16 weeks post-transplantation, flow cytometric analysis of peripheral blood revealed that KitV558Δ;T669I/+ cells were able to repopulate the recipients with greater efficiency compared to wild-type cells (Fig. 6A). A higher percentage of myeloid (Mac1+Gr1+) chimerism was observed in the peripheral blood of recipients that received KitV558Δ;T669I/+ BM cells compared to wild-type (Fig. 6B). Analysis of myeloid (Mac1+Gr1+) and B-lymphoid (B220+) lineages in the BM and spleen of primary recipients revealed significantly higher repopulation by KitV558Δ;T669I/+ than wild-type in the myeloid but not the B220+ lineage (Fig. 6C, 6D). Interestingly, HSC analysis (LSKCD34) in the BM did not reveal significantly higher KitV558Δ;T669I/+ chimerism in the primary recipients (Fig. 6E). In order to test the long-term self-renewal potential of HSC, BM cells from three primary recipient mice from wild-type or KitV558Δ;T669I/+ groups were pooled and two million or five million cells were injected into two cohorts of lethally irradiated CD45.1 mice. Flow cytometric analysis of peripheral blood to determine donor chimerism at 16 weeks revealed significantly higher donor and myeloid chimerism in both cohorts that received KitV558Δ;T669I/+ cells (Fig. 6F, 6G). Furthermore, analysis of repopulation efficiency in the BM and spleen of secondary recipients showed significantly greater donor contribution toward myeloid and B-lymphoid lineages in the recipients receiving KitV558Δ;T669I/+ cells (Fig. 6H, 6I). Interestingly, analysis of LSKCD34 HSC in the BM of secondary recipients revealed significantly higher donor contribution in the mice receiving KitV558Δ;T669I/+ cells (Fig. 6J) indicating increased long-term self renewal of KitV558Δ;T669I/+ HSC in comparison with wild-type HSC. Since the KitV558Δ;T669I/+ mice showed an increased number of LT-HSC in the spleen, we tested if the HSCs in the spleen were functional. Spleen cells (2 × 106) from wild-type or KitV558Δ;T669I/+ were mixed with CD45.1 competitor BM cells (2 × 105) and transplanted into lethally irradiated congenic recipients. At 16 weeks post-transplantation, analysis of donor chimerism in peripheral blood revealed significantly higher total, myeloid, and B-lymphoid donor contribution in recipients that were transplanted with KitV558Δ;T669I/+ spleen cells (Fig. 6K, 6L). Our results reveal that the KitV558Δ;T669I mutation confers greater advantage to stem cells in terms of self-renewal and that the HSCs in the spleen are capable of long-term self renewal.

Figure 5.

Analysis of fetal livers from wild-type and KitV558Δ;T669I/+ mice. (A): The total number of nucleated cells is higher in KitV558Δ;T669I/+ fetal livers at 14.5 dpc. (B, C): LSK frequency and numbers were not significantly different in KitV558Δ;T669I/+ mice compared to WT. (D, E): Long-term hematopoietic stem cell frequency and number were significantly higher in KitV558Δ;T669I/+ fetal livers compared to wild-type. (F): Analysis of erythroid progenitors by staining fetal livers against Ter119 and CD71 antibodies revealed significantly higher R1 progenitors in KitV558Δ;T669I/+ compared to wild-type fetal livers, while the erythroid subsets A, B, and C did not vary significantly between the two groups. (G, H): Lineage analysis revealed no significant difference in B-lymphoid (B220+), or myeloid (Gr1+) cell numbers between KitV558Δ;T669I/+ and wild-type mice. Graphs A--H: data are representative of at least two independent experiments. n = 6–8 fetal livers per group. Values are mean ± SEM. Abbreviations: FL, fetal livers; GTK, Kit(V558Δ;T669I/+); LSK, LinSca+Kit+; WT, wild type.

Figure 6.

Analysis of hematopoietic stem cell self-renewal from wild-type and KitV558Δ;T669I/+ BM and spleen. (A, B): CD45.1 mice were lethally irradiated and transplanted with CD45.1 whole BM cells (5 × 105) in competition with wild-type or KitV558Δ;T669I/+ (CD45.2) BM cells (5 × 105). Peripheral blood analyzed for donor contribution in total and myeloid compartments reveals increased donor contribution in recipients transplanted with CD45.1 and KitV558Δ;T669I/+ cell mixture (n = 8–9). (C, D): BM and spleen analyzed for CD45.2 contribution in myeloid and B-lymphoid compartments reveal higher donor contribution for the myeloid but not B-lymphoid lineage in the KitV558Δ;T669I/+ group (n = 4–6). (E): BM analyzed for donor contribution in LSKCD34 stem cell compartment did not show significant difference among the two groups (n = 5). Secondary transplantation was carried out in lethally irradiated CD45.1 recipients with two million and five million pooled whole BM cells from three donors in each primary transplanted group. (F, G): Reconstitution in peripheral blood analyzed in total and myeloid populations 16 weeks after transplantation showed higher donor contribution in both the two million and five million KitV558Δ;T669I/+ transplanted groups (n = 4–5). (H, I): CD45.2 donor contribution in BM and spleen for myeloid and B-cell subsets was also higher in the KitV558Δ;T669I/+ groups (n = 4–5). (J): Significantly higher CD45.2 contribution was observed in the LSKCD34 stem cell compartment in the BM of recipients that were injected with five million CD45.1: KitV558Δ;T669I/+ cells (n = 4–5). (K, L): CD45.1 mice were also transplanted with 2 × 106 whole spleen cells from WT or KitV558Δ;T669I/+ and cotransplanted with 2 × 105 CD45.1 whole BM cells. Reconstitution was measured in peripheral blood 16 weeks post-transplantation. Significantly higher donor contribution was observed in total, myeloid, and B-lymphoid subsets in the KitV558Δ;T669I/+ donor group n = 5 mice in each group. Data are representative of at least two independent experiments. Values in graphs A--L are mean ± SEM. Abbreviations: BM, bone marrow; GTK, Kit(V558Δ;T669I/+); LSK, LinSca+Kit+; SP, spleen; WT, wild type.

Cell Cycle Status and 5-FU Stress Response of Wild-Type and KitV558Δ;T669I/+ HSC

We next ascertained whether the oncogenic Kit mutation conferred any changes toward the proliferative rate of stem cells by analyzing BrdU incorporation in stem cells. Interestingly, BM LSKCD34 stem cells from KitV558Δ;T669I/+ mice incorporated more BrdU compared to wild-type (Fig. 7A, 7B). Although a significantly higher number of BrdU-positive stem cells was observed in the mutant BM, the difference between the wild-type and mutant was less than twofold. The self-renewal property of HSCs is linked to the cycling status of HSC, especially to cellular quiescence. In order to distinguish whether there was any difference in the quiescent G0 and resting G1 phases of the cell cycle, we stained LSKCD34 stem cells with Ki67 in conjunction with DAPI which allows distinction between G0 and G1 phases of the cell cycle. The percentage of quiescent and resting LSKCD34 stem cells in the mutant BM (Fig. 7C, 7D) or spleen (Fig. 7E, 7F) did not differ significantly from wild-type. Moreover, stem cells from the mutant and wild-type did not differ significantly in the S/G2/M phase of cell cycle. To test whether the KitV558Δ;T669I mutation provided an advantage to HSC during stress hematopoiesis, we subjected wild-type and KitV558Δ;T669I/+ mice to a single dose of 150 mg/kg 5-FU. Five-FU results in the elimination of proliferating stem and progenitor cells thereby inducing activation of the quiescent HSCs to enter the cell cycle and replenish the lost progenitors. A comparison of blood parameters showed that as expected 5-FU treatment caused a rapid decline in the WBC and neutrophil counts in both wild-type and KitV558Δ;T669I/+ mice between day 4 and day 8 of 5-FU treatment (Fig. 7G, 7H). At day 16 post-5-FU, the recovery of WBC and neutrophil counts was near baseline levels in wild-type mice in contrast to KitV558Δ;T669I/+ where an increase in their numbers above baseline was observed (Fig. 7G, 7H). Between day 4 and day 8, % HCT levels in wild-type mice also declined from an average 50% at baseline to 25%–30%, which is in accordance with our previous results [25]. In sharp contrast, % HCT levels showed only a small decline during this time frame in KitV558Δ;T669I/+ mice (Fig. 7I). In order to assess whether 5-FU induces an accelerated cycle entry in KitV558Δ;T669I/+ HSC, 5-FU-treated mice were administered BrdU and the cells from the BM were subjected to cell cycle analysis. Since it has been established previously that 5-FU treatment results in a transient loss of KIT expression on HSC and that repopulating activity is predominant in the LinSca+ population, we analyzed the LinSca+ population for changes in cell cycle status [26, 27]. While the frequency of LinSca+ cells in the BM did not change in KitV558Δ;T669I/+ compared to wild-type between day 4 and day 16 post-5-FU (data not shown), the fraction of LinSca+ cells that were BrdU positive was significantly higher in KitV558Δ;T669I/+ at day 8, and the numbers remained higher than wild-type even at day 16 (Fig. 7J). Taken together, these results then imply that 5-FU induced hematopoietic stress accelerates HSC proliferation of KitV558Δ;T669I/+ cells compared to wild-type cells.

Figure 7.

Stem cell proliferation in wild-type and KitV558Δ;T669I/+ BM and spleen, and response to 5-FU. (A): Stem cell turnover was measured in the BM by BrdU incorporation. Graphical representation of percentage of BrdU+ BM LSKCD34 stem cells in mutant mice. Values are mean ± SEM; n = 7 mice in each group. (B): Representative BrdU staining profile of LSKCD34 stem cells from wild-type and mutant BM. (C): BM LSKCD34 stem cells in G0 (quiescent), G1 (resting), and S/G2/M (proliferating) phases of cell cycle as measured by Ki67 and DAPI staining. (D): Representative staining profile of LSKCD34 cells from wild-type and mutant BM stained with Ki67 and DAPI. (E): Spleen LSKCD34 stem cells from wild-type and mutant in G0, G1, and S/G2/M phases of cells cycle measured by Ki67 staining. (F): Representative staining profile of LSKCD34 cells from wild-type and mutant spleen stained with Ki67 and DAPI. Values in C and E are mean ± SEM; n = 5 mice in each group. Data are representative of at least three independent experiments. (G, H): A single dose of 150 mg/kg b.wt. 5-FU led to reduced WBC and neutrophil numbers in both wild-type and mutant at day 8 of 5-FU treatment followed by a recovery in numbers by day 16. (I): Hematocrit did not fall sharply in KitV558Δ;T669I/+; however, in the wild-type hematocrit declined sharply at day 8 after treatment. (J): 5-FU-treated wild-type and mutant mice were administered BrdU 1 day before each analysis and BM was harvested to analyze BrdU incorporation in LinSca+ cells by flow cytometry. At day 8, the number of cycling cells is higher in the KitV558Δ;T669I/+ mice compared to wild-type. Values are mean ± SEM. n = at least three mice in each group per time point. Abbreviations: 5-FU, 5-fluorouracil; BM, bone marrow; DAPI, 4′,6-diamidino-2-phenylindole; GTK, Kit(V558Δ;T669I/+); HCT, hematocrit; LSK, LinSca+Kit+; SP, spleen; WT, wild type.

Discussion

A hallmark of KIT-KITL signaling is its role in the erythroid lineage evident in the form of macrocytic anemia which is observed in mice with Kit loss-of-function mutations. Recently in mice carrying the KitV558Δ;T669I/+ gain of function mutation, we showed that the HCT was elevated due to increased BFU-E progenitors that were hypersensitive to KITL concentrations [11]. In accordance with increased numbers of BFU-E, the numbers of erythroid progenitors assessed in this study were also increased in the spleen of KitV558Δ;T669I/+ mice and greater frequency of progenitors were in cell cycle in the KitV558Δ;T669I/+ spleen than in the KitV558Δ;T669I/+ BM. In order to test whether the spleen was essential for the extramedullary erythropoiesis observed in the KitV558Δ;T669I/+ mice, we performed splenectomy. Splenectomy normalized HCT in the KitV558Δ;T669I/+ mice to near wild-type levels suggesting a highly significant contribution of the spleen toward maintenance of high erythroid output in KitV558Δ;T669I/+ mice. Similar results have been obtained in JAK2V617F mutant mice where splenectomy resulted in normalizing HCT levels to wild-type levels and in EPO transgenic mice where splenectomy substantially reduced HCT levels [17, 28].

KIT has an important role in the expansion of the cells of the myeloid lineage especially mast cells. But unlike mast cells, where the role of KIT in their expansion and survival has been well documented, the importance of KIT in the expansion of cells of other myeloid lineages or their precursors is not well-defined. BAC transgenic mice with a KitD816V mutation develop severe mastocytosis have increased erythroid cell numbers and develop B-cell tumors yet these mice did not have increased myeloid cell numbers [9]. Interestingly, in many patients, the KitD816V mutation was detected not only in mast cells but also in earlier hematopoietic progenitor cells and other myeloid cells indicating that this mutation may be conferring proliferative capacity not only to mast cells but also to myeloid progenitors [29]. Notably, KitV558Δ;T669I/+ mice have increased granulocyte and monocyte numbers in the BM and spleen. Earlier work measuring CMP numbers based on CFU-S assays had shown that in Kit loss-of-function mutants such as Wv/+, W42/+, and W41/+, CMP numbers were reduced suggesting that Kit signaling may be important for proliferation of myeloid progenitors [2]. While we did not see changes in CMP or GMP or CFU-S colony numbers in the BM of KitV558Δ;T669I/+ mice, in accordance with the increased erythroid numbers, MEPs were significantly increased in the BM of mutant mice compared to wild-type. Our results compare with the PV mouse model where MEP numbers were significantly increased in the BM but not CMP or GMP [30, 31]. While Kit has a role in B-cell maturation [32] and a profound effect on B-cell proliferation was observed in BAC transgenic KitD816V mice with the development of B-cell tumors [9], B-cell and T-cell numbers in KitV558Δ;T669I/+ mice were not statistically different from wild-type. The effects observed on the erythroid and myeloid cells suggest that the myeloerythroid lineage is preferably affected in KitV558Δ;T669I/+ mice.

The changes in multipotential progenitor cells (LSK) or stem cell (LT-HSC) numbers were minimal in the BM of KitV558Δ;T669I/+ mice compared to wild-type. In contrast, LSK and LT-HSC were 11-fold and 6-fold increased in the spleen of KitV558Δ;T669I/+ mice compared to wild-type, respectively. Additionally, stem cells were found in circulation. The presence of LT-HSC in the peripheral blood or spleen has been observed in other myeloproliferative mouse models such as the RbΔ/Δ mice, where the BM environment is altered due to myelofibrosis, thereby favoring the mobilization of hematopoietic cells including LT-HSC from the marrow to the spleen [33]. Since the marrow of KitV558Δ;T669I/+ mice did not turn fibrotic as the mice aged [11], it is possible that a combination of constrained space and cell-intrinsic factors in the marrow forced the LT-HSC into the spleen. The requirement of HSC for KITL increases during ontogeny, such that adult HSCs require more KITL than their fetal counterparts [21, 22]. Hence, we assessed if increased KIT signaling in the KitV558Δ;T669I/+ mice had any effect on fetal HSC numbers. While FLs of KitV558Δ;T669I/+ mice had similar numbers of LSK progenitor cells compared to wild-type, LT-HSC numbers were increased more than twofold compared to wild-type in agreement with a reduced threshold for KIT signaling in the FL.

KIT has been shown to be important for self-renewal of LT-HSC. The BM from W41/W41 mice or W42/+ are deficient in long-term generation of hematopoiesis in recipient BM [2, 34, 35] and interestingly long-term but not short-term competitive activity of W41/W41 FL cells was also found to be defective [36]. Evaluation of the reconstitution ability of KitV558Δ;T669I/+ HSCs by competitive repopulation assays showed that donor contribution toward the myeloid lineages was higher with the KitV558Δ;T669I/+ donors compared to wild-type in primary and secondary transplantations. Notably, while the donor-derived stem cell numbers were similar for wild-type and KitV558Δ;T669I/+ donors after primary transplantation, the contribution of donor-derived stem cells increased in KitV558Δ;T669I/+ donors following secondary transplantation, indicating increased self-renewal capacity of KitV558Δ;T669I/+-derived donor LT-HSC. The presence of significant numbers of LT-HSCs in the spleen of KitV558Δ;T669I/+ mice prompted us to test whether they were functional [16]. Interestingly, transplanting CD45.1 recipients with wild-type or KitV558Δ;T669I/+ spleen cells in competition with congenic BM cells revealed significant spleen-derived multilineage activity at 16 weeks, showing that the KitV558Δ;T669I/+ mice have functional HSC in the spleen. While the precise role of the HSC in the spleen in general is unclear, it has been suggested that the spleen may function as a storehouse of HSC to rapidly generate erythroid cells in conditions of stress [16]. The increased self-renewal of HSC in our gain-of-function mutant supports earlier work which showed that KITL greatly enhances self-renewal ability of both fetal and adult stem cells, despite their different sensitivity to this factor [21]. While we observed higher BrdU incorporation in the KitV558Δ;T669I/+ stem cells, we did not observe significantly decreased quiescence or increased S/G2/M phase of KitV558Δ;T669I/+ BM and spleen HSC in comparison to wild-type, indicating that the effect of Kit mutation on self-renewal is perhaps greater than its effect on proliferation of stem cells. 5-FU is known to deplete cycling HSCs and progenitors while sparing resting LT-HSCs due to their slower cycling characteristics [37]. These cells then expand and regenerate the depleted progenitors. Since the KitV558Δ;T669I/+ LT-HSC had increased self-renewal capacity based on competitive repopulation properties, we tested if upon 5-FU treatment, these cells would show an accelerated response to stress. We saw increased expansion of WBC and neutrophils in the 5-FU treated KitV558Δ;T669I/+ mice indicating rapid expansion of progenitors in these mice compared to wild-type. Furthermore, the percentage of HSC in cycle was also increased.

Conclusion

In conclusion, our analysis of the hematopoietic characteristics of the KitV558Δ;T669I/+ mice shows that the Kit gain-of-function mutation has a significant effect on the proliferation of the myelo-erythroid cell lineage both in the BM and the spleen, which ultimately results in elevated HCT and microcytosis. While there is an expansion of LT-HSCs especially in the spleen of KitV558Δ;T669I/+ mice, overall the Kit gain-of-function mutation has an important role in regulating the self-renewal of LT-HSC.

Acknowledgements

We thank Dr. Jae-Hung Shieh and Ling Bo Shen for expert assistance with hematological determinations. We also thank Adriana Guevara and Jun-Yuan Tan for technical assistance. We would like to thank Dr. Joseph M. Scandura for helpful suggestions. Special thanks to Drs. Jan Hendrikx and Jennifer Wilshire for their help and discussion related to FACS analysis. We thank Antoinette Rookard, Dr. Peter Romanienko, and members of RARC for excellent management of mice. We thank the veterinary service of MSKCC for help with splenectomy of mice. We furthermore acknowledge the assistance of the members of Antitumor Assessment Facility, especially Dr. Elisa de Stanchina and Huiyong Zhao. We also would like to thank John Burrowes for secretarial assistance. This work was supported by NIH Grants RO1-HL55748, R01-CA102774, and P50-CA140146 and the Starr Cancer Consortium.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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