Quantitative effects of Nf1 inactivation on in vivo hematopoiesis

The NF1 tumor-suppressor gene is frequently inactivated in juvenile myelomonocytic leukemia, and Nf1 mutant mice model this myeloproliferative disorder (MPD). Competitive repopulation assays were performed to quantify the proliferative advantage of Nf1^–/– hematopoietic cells in vivo. Nf1 mutant stem cells demonstrated a growth advantage that was greatest in myeloid lineage cells and least pronounced in T lymphocytes. Surprisingly, although low numbers of Nf1-deficient cells consistently outcompeted wild-type cells, levels of chimerism were stable over months of observation, and MPD was not observed unless threshold numbers of mutant cells were injected. These data showing that normal competitor cells can strongly modulate the growth of mutant populations in vivo have general implications for modeling cancer in the mouse. In particular, strains in which cancer-associated mutations are expressed in fields of target cells may not accurately model early events in tumorigenesis because they eliminate the requirement for a mutant clone to outcompete resident normal cells.

Cancer results from a discrete series of genetic alterations in a susceptible target cell, which perturb fundamental properties such as proliferation, differentiation, survival, and responsiveness to growth-promoting or inhibitory cytokines (1). The biochemical activities of proteins implicated in tumorigenesis have largely been elucidated by a combination of in vitro experiments using purified peptides and by in vivo studies in which recombinant cDNAs encoding genes of interest are overexpressed in cultured cell lines. While powerful, these methodologies neither address how individual genetic lesions behave in the context of a primary cell at a specific stage of differentiation, nor do they provide quantitative data comparing the growth of mutant cells versus their normal counterparts. These questions, which have major implications both for characterizing early events in tumorigenesis and for designing rational new cancer therapies, can now be studied using cells derived from mice engineered to carry specific cancer-associated genetic lesions.

Mutations in the NF1 tumor-suppressor gene cause neurofibromatosis type 1 (NF1), a common dominantly inherited disorder. NF1 encodes neurofibromin, which binds to p21^Ras (Ras) family members with high affinity and downregulates signaling by acting as a GTP-activating protein for Ras (2, 3). Individuals with NF1 are predisposed to certain cancers, including malignant peripheral nerve sheath tumor, pheochromocytoma, astrocytoma, and juvenile myelomonocytic leukemia (JMML) (4). JMML is a relentless myeloproliferative disorder (MPD) of young children characterized by overproduction of myeloid lineage cells that infiltrate hematopoietic and nonhematopoietic tissues (5, 6).

Homozygous Nf1-deficient (Nf1^–/–) mice succumb at embryonic day 12.5–13.5 (E12.5–13.5) from complex cardiac defects (7, 8); however, Nf1^–/– fetal liver cells efficiently rescue hematopoiesis in irradiated hosts (9–11). These recipients consistently develop a JMML-like MPD with elevated myeloid cell counts, splenomegaly, and infiltration of myeloid cells into the liver and spleen (9–11). Genetic and biochemical studies of human and murine hematopoietic cells have shown that Nf1 functions as a tumor suppressor through the ability of neurofibromin to negatively regulate Ras output (9, 12–15). Hypersensitivity to GM-CSF plays a central role in the aberrant growth of human JMML and murine Nf1-deficient cells (6, 9, 14). In addition, Nf1-deficient cells form excessive numbers of progenitor colonies in response to stem cell factor (SCF), and recent studies have demonstrated abnormal signaling from its receptor, the c-kit tyrosine kinase, in heterozygous Nf1 mutant mast cells (10, 16, 17).

While much has been learned about how inactivation of Nf1 deregulates hematopoietic growth, important questions remain. First, the quantitative effects of Nf1 loss on the growth of hematopoietic cells are unknown. Second, although adoptive transfer studies in mice suggest that inactivation of Nf1 is both necessary and sufficient to induce MPD in irradiated recipients, these studies do not account for the potential effects of coexisting normal cells on the growth of Nf1^–/– stem cells within the marrow microenvironment. Finally, data from patients with JMML suggest that the disease originates in a cell with the capacity to differentiate into myeloid or lymphoid progeny in at least some instances (18, 19). This is certainly true in murine recipients repopulated with Nf1^–/– fetal liver cells, in which the entire hematopoietic system is derived from mutant cells. Despite this, the hematologic disorders that arise in children with NF1 and in murine recipients of Nf1^–/– fetal liver cells are almost invariably myeloid. These observations suggest a differential requirement for neurofibromin in myeloid versus lymphoid growth control, and it remains unclear whether neurofibromin modulates p21^ras GTPase activity in other hematopoietic lineages (20, 21).

The hematopoietic system is hierarchical in nature, well-established in vitro assays exist for measuring the growth of precursors derived from discrete compartments, and the technique of competitive repopulation permits direct in vivo comparisons between mutant and wild-type cells in a variety of lineages. Hematopoiesis therefore provides a model experimental system for quantifying the effects of specific cancer-associated mutations such as Nf1 inactivation in differentiating primary cells. We performed competitive repopulation studies to compare the growth of Nf1^–/– hematopoietic cells to that of normal cells in vivo. We hypothesized that Nf1 mutant cells would consistently outcompete wild-type cells and that this growth advantage would increase over time such that all recipients engrafted with mutant cells would ultimately develop MPD. We further hypothesized that inactivation of Nf1 would confer a strong proliferative advantage in myeloid cells, and we set out to determine whether this was also seen in the stem cell compartment and in lymphoid cells.

Here we show that the genetic background of Nf1-deficient donor cells influences the phenotype of the MPD that develops after adoptive transfer. Furthermore, inactivation of Nf1 results in differential expansion of immature progenitors in the spleens and bone marrows of irradiated recipients. We also find that Nf1-deficient cells demonstrate a relatively modest growth advantage over normal cells that is more pronounced in, but is not restricted to, the myeloid compartment. Surprisingly, Nf1-deficient cells did not progressively outcompete their wild-type counterparts over months of observation. Instead, we found stable chimerism in irradiated recipients. Furthermore, MPD only developed in recipients that received threshold numbers of Nf1 mutant cells. These data suggest that genetically engineered murine cancer models that create fields of mutant cells may not recapitulate all of the mutational events that are required for clonal outgrowth from a single mutant precursor.

Effects of strain background on MPD. We crossed the Nf1 mutation onto a C57BL/6 background for 10 generations in order to perform competitive repopulation experiments in congenic mice (see below). Unexpectedly, the background of the donor fetal liver cells modulated the phenotype of the MPD in irradiated hosts. Syngeneic recipients transplanted with 129/Sv Nf1^–/– fetal liver cells had modestly elevated total leukocyte and myeloid cell counts 2 months after adoptive transfer and showed marked increases by 4 months (Figure 1a). Recipients of C57BL/6 Nf1^–/– cells showed lower blood counts at these times (Figure 1a). The genetic background of donor fetal liver cells had more striking effects on survival and splenomegaly. Mice transplanted with 129/Sv cells showed substantial mortality by 6 months (Figure 1b); this was associated with splenomegaly (Figure 1c) and with myeloid cell infiltration within the liver and spleen (data not shown). Although recipients of Nf1^–/– C57BL/6 fetal liver cells uniformly developed MPD, the course was more indolent with enhanced survival and smaller spleen size (Figure 1, b and c). Approximately 90% of these recipients survived to at least 1 year of age (data not shown). Heterozygous Nf1 mutant 129/Sv and C57BL/6 mice were mated to ascertain which hematopoietic phenotype is dominant. Recipients injected with F₁ fetal liver cells developed markedly elevated total peripheral white blood cell and myeloid cell counts (Figure 1d), prominent splenomegaly, and early mortality that was at least as severe as the MPD seen in the inbred 129/Sv background. Together, the results presented in Figure 1 indicate that crossing the Nf1 mutation onto the C57BL/6 background attenuates the MPD that arises in recipients of Nf1-deficient fetal liver cells.

Figure 1

Leukocyte counts, spleen sizes, and survival in recipients of Nf1^–/– fetal liver cells from C57BL/6, 129/Sv, or F₁ embryos. (a and b) Recipients injected with wild-type C57BL/6 fetal liver cells are denoted by open squares, and recipients injected with Nf1^–/– C57BL/6 cells are denoted by filled squares. Recipients that received wild-type or Nf1^–/– 129/Sv fetal liver cells are denoted by open and filled triangles, respectively. Leukocyte counts and percentage of survival 2–6 months after adoptive transfer. (c) Spleen weights at sacrifice. Filled bars represent C57BL/6. Open bars represent 129/Sv. *P < 0.05 comparing ^–/– to ^+/+ spleen weight within the same donor strain. (d) Total leukocyte (WBC) and myeloid cell counts of recipient mice reconstituted with either Nf1^–/– (squares) or wild-type (circles) fetal liver cells from F₁ 129/Sv × C57BL/6 embryos. Recipient strain had no effect on WBC counts, survival, or spleen size (data not shown). WBC, white blood cell.

Organ-specific expansion of immature Nf1-deficient progenitors in vivo. Nf1^–/– fetal liver cells form increased numbers of colonies derived from both primitive progenitors with significant replating potential, referred to as HPP-CFC, and from more mature lineage restricted myeloid progenitors called LPP-CFC (10). Since splenomegaly is a significant component of both JMML and of the MPD seen in mice transplanted with Nf1^–/– cells, we compared LPP-CFC and HPP-CFC numbers in the bone marrows and spleens of recipients of C57BL/6 fetal liver cells. Absolute numbers of progenitors per organ were calculated by multiplying the frequency of each progenitor detected in two-layer agar cultures by the number of low-density cells per organ. The bone marrows of mice that received Nf1^–/– cells showed a marked increase in the numbers of LPP-CFC progenitor cells compared with recipients of wild-type cells, while absolute numbers of more primitive hematopoietic HPP-CFC progenitors were equivalent (Figure 2). Thus, expansion of Nf1^–/– cells occurs at the level of relatively mature and differentiated progenitors in recipient marrow. In contrast, there was marked expansion of both HPP-CFC and LPP-CFC compartments in the spleens of mice transplanted with Nf1^–/– cells. The bone marrows and spleens of recipients engrafted with Nf1^–/– fetal liver cells also showed a marked expansion in the numbers of burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte, macrophage, erythroid, and megakaryocyte (CFU-GEMM) progenitors (data not shown).

Figure 2

LPP and HPP progenitors in bone marrow and spleen. Growth of LPP and HPP colonies from the bone marrows and spleens of recipients transplanted with Nf1^–/– (filled bars) or with wild-type (open bars) fetal liver cells. Total numbers of LPP-CFCs and HPP-CFCs per organ are shown. *P < 0.01 comparing Nf1^–/– vs. Nf1^+/+.

Quantitative growth advantage of Nf1^–/– hematopoietic cells. The competitive repopulation assay provides a way of quantifying the effects of a specific mutation on the growth of cells within multiple hematopoietic compartments (23). This assay is sensitive to small differences in test cell populations because the entire differentiation pathway is analyzed. FACS was performed on fetal liver cells from Nf1^–/– and Nf1^+/+ littermates to isolate Sca1⁺lin^–/dim cells, a fraction that is highly enriched for stem and progenitor cells. A range of test cell numbers of each genotype was mixed with 5 × 10⁵ congenic competitor cells from a strain (C57BL/6 BoyJ) that expresses an isoantigen of the pan-hematopoietic cell surface marker CD45. These cells were transferred into irradiated hosts, and sequential peripheral blood samples collected from these recipients over 6 months were analyzed by FACS. As expected, the total CD45.1 (BoyJ) and CD45.2 (test cell) chimerism of individual animals approximated 100% (100% ± 1%, data not shown). Recipients transplanted with Nf1^–/– fetal liver cells had a two- to threefold higher test cell chimerism over a 100-fold range of input test cells relative to wild-type cells (Figure 3a). To determine which lineages are affected by inactivation of Nf1, we performed two-color staining using anti-CD45.2 and a series of Ab’s that are specific to myeloid or lymphoid lineages. Although recipients engrafted with Nf1-deficient fetal liver cells consistently maintain normal lymphocyte counts and do not develop lymphoid malignancies (9–11), Nf1^–/– cells achieved a growth advantage in both myeloid and lymphoid lineages (Figure 3, b–e). However, there were quantitative differences in the proliferative advantage of Nf1^–/– cells between myeloid and lymphoid compartments. For example, while adoptive transfer of 250 Sca1⁺lin^–/dim cells resulted in approximately 50% chimerism in granulocytes and macrophages, equivalent chimerism in T lymphocytes required transplantation of 1,000 Sca1⁺lin^–/dim cells (Figure 3, b–d). Nf1^–/– B lymphocytes had a proliferation advantage over Nf1^+/+ cells that was similar overall to the advantage observed in the myeloid lineages (Figure 3e). Serial analyses using an established measure of repopulating ability (24) demonstrated that the proliferative advantage of Nf1-deficient cells was stable over 6 months of observation (Figure 3f). Finally and importantly, there was a clear threshold effect with respect to the number of Nf1^–/– Sca1⁺lin^–/dim fetal liver cells transferred and the development of MPD. Recipients that received less than 1,000 test cells in four independent experiments maintained normal leukocyte and myeloid cell counts during 6 months of observation (Figure 3g) and did not develop enlarged spleens nor have evidence of disease. By contrast, recipients injected with 1,000–5,000 Nf1-deficient test cells consistently developed overt MPD with elevated leukocyte counts and an increased percentage of myeloid cells (Figure 3g). These mice also developed marked splenomegaly (mean spleen weight 310 ± 68 mg SEM) and showed reduced survival comparable to recipients transplanted with Nf1^–/– cells only.

Figure 3

Comparative growth of Nf1^–/– and wild-type Sca1⁺lin^–/dim cells in competitive repopulation assays. Nf1^–/– cells are shown as squares and wild-type cells as circles. (a–e) Test cell chimerism in blood leukocytes (CD45.2-positive), monocytes and macrophages (Mac1-positive), granulocytes (Gr1-positive), T lymphocytes (CD3-positive), and B lymphocytes (B220-positive). *P < 0.05 comparing Nf1^–/– vs. Nf1^+/+. (f) Comparative repopulating abilities of Nf1^–/– and wild-type cells over time. (g) Evaluation of the proportion of myeloid and lymphoid cells in recipients reconstituted with 100–5,000 Sca1⁺lin^–/dim test cells.

Enhanced growth of Nf1^–/– cells after secondary transfer. The observation that Nf1^–/– cells outcompete their wild-type counterparts in all hematopoietic lineages provided indirect evidence of a growth advantage within the stem cell compartment. To test this idea further, bone marrow cells from primary recipients of fetal liver cell transplants showing a 50% level of chimerism in blood leukocytes were transferred into secondary hosts, and chimerism was assessed 4 months later. A stable level of CD45.1/CD45.2 chimerism was observed when cells from primary hosts that had received test and competitor cells collected from wild-type fetal livers were transferred. In contrast, secondary recipients transplanted with bone marrow cells from primary hosts that had initially received Nf1-deficient test cells consistently showed a substantial increase in the percentage of CD45.2-positive leukocytes derived from Nf1^–/– donor cells (Figure 4). Thus, loss of Nf1 results in an increase in stem cell repopulating ability.

Figure 4

Chimerism of secondary recipients. Total test cell chimerism (a) and lineage-specific chimerism (b) is shown for recipients that received bone marrow cells from primary recipients showing 50:50 CD45.1/45.2 chimerism. Mice were transplanted from primary recipients that received Nf1^–/– (filled bars) or wild-type (open bars) fetal liver test cells, respectively.

Adoptive transfer of Nf1-deficient fetal liver cells into irradiated recipients provides a model of JMML (9) that has been used for biologic and preclinical studies (10, 11, 25). One finding from this study is that the most immature population of progenitors assayed, the HPP-CFC, was markedly expanded in the spleens of recipient mice but is normal in the marrow. This suggests that the marrow microenvironment does not support expansion of the most immature Nf1^–/– progenitors, while the spleen is permissive for overgrowth in the HPP-CFC compartment. These data may be relevant to the pathogenesis of human JMML, which is frequently characterized by massive splenomegaly (5, 6).

We found that crossing the Nf1 mutation onto a C57BL/6 background modulated the phenotype associated with gene inactivation. Data from the F₁ cross between C57BL/6 and 129/Sv revealed that the 129/Sv background dominantly enhances the severity of the MPD caused by inactivation of Nf1. An F₂ cross will be required to ascertain if this is encoded by a single locus or is due to a number of interacting modifiers. Interestingly, male sex and maternal transmission of a mutant NF1 allele influence the risk of developing myeloid leukemia in children with NF1 (26, 27). Our data, which underscore the importance of accounting for genetic background in characterizing murine models of human cancer, are consistent with a recent study in which strain background modified the incidence of glioblastoma in a line of mice that carried mutations of both Nf1 and p53 in cis (28).

While the competitive repopulation assay is commonly viewed as a sensitive way of measuring the long-term proliferative potential of hematopoietic stem cells, our data indicate that the growth advantage of mutant cells varies in a lineage-specific manner. Inactivation of Nf1 confers a growth advantage that is most pronounced in the myeloid compartment and less apparent in T lymphocytes. We also performed secondary adoptive transfer experiments to prove that that Nf1^–/– stem cells outcompete wild-type cells. Our data are consistent with previous studies showing that both children with NF1 and heterozygous Nf1 mutant mice are strongly predisposed to myeloid, but not lymphoid, malignancies (9, 26, 29, 30). Data from our laboratories and others implicate differential responses of the progeny of mutant stem cells to extracellular stimuli such as growth factors, which confer a growth (9, 10) and survival (16, 31) advantage to Nf1 mutant hematopoietic cells. These cellular phenotypes might be due to an alteration in the threshold responsiveness of cells to basal levels of cytokine growth factors that signal through p21^ras -GTP (17). Expression of the BCR-ABL fusion oncogene and inactivation of the tumor suppressor Pten are thought to perturb growth, in part, by sensitizing cells to growth factors (32, 33). Furthermore, hyper-responsiveness to GM-CSF and, perhaps, to SCF and other myeloid growth factors is implicated as playing a central role in human JMML and in the MPD that develops in recipients of Nf1^–/– transplants (6, 11, 34). In vitro data indicate that Nf1-deficient hematopoietic cells have increased proliferation and survival in response to multiple growth factors. While mice transplanted with only Nf1^–/– fetal liver cells invariably develop MPD, and Nf1^–/– cells outcompete wild-type cells in competitive repopulation experiments, it is unclear whether in vivo expansion of myeloid cells is secondary to increased proliferation and/or survival. Preliminary data, based on FACS analysis of phenotypically identified progenitor/stem cell populations, indicate that there are more Nf1^–/– cells in S-phase as compared with wild-type cells and that Nf1^–/– cells are significantly more resistant to apoptosis (unpublished results, D. Ingram and W. Clapp). Studies are currently underway to determine the biochemical mechanisms for these phenotypes and to determine the relative role of each of these cellular functions in contributing to the myeloproliferative disease observed in mice transplanted with Nf1^–/– cells.

We were surprised to discover that low numbers of Nf1-deficient Sca1⁺lin^–/dim engrafted readily in the stem cell compartment and achieved a growth advantage over wild-type cells in all differentiated lineages tested, but this did not invariably evolve to MPD. Instead, threshold numbers of Nf1^–/– cells were required to induce the JMML-like myeloid disorder. The stable levels of chimerism observed over 6 months of observation are unlikely to be explained by a slow rate of stem cell turnover because murine stem cells replicate approximately once every 2.5 weeks in vivo (35). Instead, our data imply that a steady state is achieved between Nf1-deficient and wild-type cells in recipient bone marrows. This may be similar to human patients who have been successfully treated for myeloid leukemia associated with a translocation t (8;21), many of whom continue to express a leukemia-associated fusion mRNA for many years (36). The capacity of normal cells within the marrow microenvironment to modulate the fate of mutant precursors was shown recently in a study in which an intrinsic defect erythroid differentiation in cells overexpressing the GATA-1 transcription factor was rescued in a cell nonautonomous manner (37). We have shown recently that heterozygous inactivation of Nf1 rescues many of the phenotypic and biochemical defects found in White spotted locus (W)⁴¹ mutant mast cells (16). Similarly, it will be interesting to determine if haploin sufficiency for Nf1 confers a growth advantage in hematopoietic stem cells.

The absence of MPD in mice that received fewer than 1,000 Nf1^–/– Sca1⁺lin^–/dim fetal liver cells implies that cooperating mutations might be required for induction of MPD in a marrow microenvironment that is mostly comprised of wild-type cells. This idea is consistent with the relatively long latency before the onset of myeloid malignancy in untreated and cyclophosphamide-exposed heterozygous Nf1 mutant mice (8, 25). Furthermore, somatic inactivation of Nf1 was not invariably associated with evidence of MPD in the cyclophosphamide study (25). These data are also compatible with human data showing that cooperating genetic events such as loss of chromosome 7 are often found in addition to inactivation of NF1 in the bone marrows of children who develop myeloid malignancies (26, 38, 39).

Murine cancer models frequently rely on either overexpressing an oncogene product or deleting a tumor suppressor in a field of target cells. How accurately this reflects human tumorigenesis, which is generally thought to involve the sequential acquisition of mutations in progeny derived from a single mutant precursor cell, is unknown. In particular, the potential inhibitory effect of normal cells within tissue microenvironments on the proliferation of mutant cells has received limited attention. We have shown that inactivating Nf1 in hematopoietic stem cells only results in MPD if threshold numbers of mutant cells are injected. These data infer that tumor initiation and clonal outgrowth are distinct events (Figure 5). In leukemias that arise spontaneously from a single aberrant precursor, it is likely that cooperating genetic lesions are required for clonal dominance (Figure 5). Our data suggest that transgenic models in which tumors derived from fields of mutant cells may obviate the requirement for one or more early cooperating genetic lesions that facilitate clonal outgrowth in vivo. If this is true, caution should be exercised in extrapolating data regarding tumor biology and preclinical responses to experimental therapeutics from these model systems to human cancers.

Figure 5

Proposed model for spontaneous development of MPD/JMML. Loss of the normal Nf1 allele in an early hematopoietic cell (dark cell) initiates leukemia development. One (or more) cooperating genetic event is required to permit this clone to outcompete normal stem cells in the marrow microenvironment. This mutant population induces clinical disease, which is driven by aberrant growth in response to GM-CSF and to other myeloid growth factors that signal through Ras.

We are indebted to Tyler Jacks for providing Nf1 mice and for valuable discussions. We also thank Mervin Yoder and Laura Haneline for their comments during the preparation of the manuscript. This work was supported by American Cancer Society grant LBC-98582, by FIRST award R29 CA74177 to D.W. Clapp, and by R01 CA72624 to K. Shannon.

Youyan Zhang and Brigit R. Taylor contributed equally to this work.

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