Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells

Abstract

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis, and mutations in CBL have been identified in myeloid malignancies. Here we show that, in contrast to Cbl or Cbl-b single-deficient mice, concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl, Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines, and show altered biochemical response to thrombopoietin. Thus, Cbl and Cbl-b play redundant but essential roles in HSC regulation, whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies.

Members of the Casitas B-cell lymphoma (Cbl) protein family are critical negative regulators of protein tyrosine kinase (PTK)-mediated signal transduction pathways (1–3). An N-terminal tyrosine kinase binding (TKB) domain, composed of a four-helical bundle, a calcium-binding EF hand, and a variant SH2 domain, mediates specific docking of Cbl proteins on activated PTKs (and some nonkinase components of PTK signaling pathways) through cognate phosphotyrosine-containing motifs. A short linker region and a RING finger domain immediately C-terminal to the TKB domain mediate interaction with E2 ubiquitin-conjugating enzymes and are essential for E3 activity of Cbl proteins.

In the past few years, several groups have reported mutations of the CBL gene in a subset of patients with myeloid neoplasms (4–13). A large proportion of CBL mutations is associated with myelodysplastic syndrome/myeloproliferative disorder (MDS/MPD), a heterogeneous group of hematopoietic malignancies characterized by deregulated hematopoiesis and a high propensity to develop acute myeloid leukemia (AML). Strikingly, CBL mutations have been identified in more than 10% of patients with juvenile myelomonocytic leukemia (JMML), a pediatric subtype of MDS/MPD with excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). In both adult and pediatric cases, a majority of the CBL mutations cluster within the linker and RING finger domains. Interestingly, only rare CBLB mutations have been detected in these studies, although not all studies have looked for such mutations.

Why mutations in CBL are specifically associated with MDS/MPD and how these mutations produce the disease are of obvious interest. A recent study has demonstrated that Cbl protein functions to limit the size of the hematopoietic stem cell (HSC) compartment, and that Cbl-null mice show an expansion of HSCs with an enhanced ability to mediate long-term bone marrow repopulation (14). However, Cbl-null animals develop only mild, nonlethal MPD, indicating that other factors are involved. A unique feature of patient-derived CBL mutations is their frequent association with uniparental isodisomy at 11q23, where the CBL gene resides, resulting in loss of the wild-type CBL allele and duplication of the mutant allele. This suggests that mutant Cbl proteins may possess a gain-of-function phenotype that confers selective advantage to neoplastic cells. Additionally, it has been suggested that wild-type Cbl may compete with mutant proteins and that increased dosage of mutant Cbl proteins in neoplastic cells may counter this inhibition (11). Consistent with these propositions, the leukemia-associated Cbl mutants were more transforming in Cbl-null HSCs compared with wild-type HSCs (11). Lack of lethal MDS/MPD in Cbl-null mice also suggests that Cbl and Cbl-b protein may function in a redundant manner in regulating HSCs and that duplication of the mutant CBL allele in leukemic patients may also reflect a need to counter the effect of Cbl-b. Here we report that mice with concurrent deficiency of Cbl and Cbl-b in the HSC compartment succumb to aggressive MPD at a young age. These animals exhibit a marked expansion of HSCs in bone marrow that can transfer MPD to recipient animals. These studies demonstrate a redundant yet essential functional role of Cbl and Cbl-b in HSC regulation and myelopoiesis, and provide a model to investigate the mechanisms by which aberrations of Cbl proteins produce myeloid lineage disorders.

Results

MMTV-Cre; Cbl^flox/flox; Cblb^del/del Mice Develop an Aggressive, Fully Penetrant MPD at an Early Age.

The original intent of mouse crosses described here was to investigate the role of Cbl-family proteins in mammary gland development and homeostasis. As mice with germ-line deletion of both Cbl and Cbl-b show early embryonic lethality, we crossed mice with a conditional allele of Cbl (Cbl flox allele) (15) and a null allele of Cblb (Cblb del allele) (16) to a mammary gland-targeting Cre transgenic mouse strain, MMTV-Cre, where the expression of the Cre recombinase is directed from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter (17). In this model, combined deficiency of Cbl and Cbl-b is expected in tissues where MMTV-Cre is active on a general Cbl-b-deficient background.

Notably, MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice were born at a sub-Mendelian ratio (36 out of 220 in MMTV-Cre; Cbl^flox/flox; Cblb^del/+ to Cbl^flox/flox; Cblb^del/del crosses, where 1 out of 4 offspring was expected to be MMTV-Cre; Cbl^flox/flox; Cblb^del/del) but developed normally until weaning age. However, both male and female MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice started to show signs of distress including hunched posture, unkept fur, and reduced locomotion at around 5 wk of age, and most of them either died or had to be euthanized for humane reasons by 8 wk of age (Fig. 1A). There was no statistically significant difference in body weight between age- and sex-matched controls and MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice. No tumors or abnormal bleeding were observed. Median survival time for MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice was 67 d. Littermates of other genotypes appeared healthy up to 300 d of age.

Fig. 1.

MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice develop fatal MPD. (A) Kaplan–Meier survival analysis of a cohort of MMTV-Cre; Cbl^flox/flox; Cblb^del/del (dKO) mice (n = 33) and mice with other genotypes (Other; MMTV-Cre; Cbl^flox/flox; Cblb^del/+ mice, Cbl^flox/flox; Cblb^del/del mice, and Cbl^flox/flox; Cblb^del/+ mice; total n = 266). The difference in survival between the two groups was significant by log-rank test, P < 0.0001. (B) Histopathology of MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice. Myeloid infiltration is observed in the white pulp of the spleen (top panels) and in the liver (bottom panels). [Scale bars, 500 μm (Upper) and 100 μm (Lower).] (C) Peripheral leukocyte count and differential analysis. The leukocyte count was significantly elevated in MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice compared with mice with other genotypes (P < 0.0001 by unpaired, two-tailed t test). L, lymphocytes; M, monocytes; N, neutrophils; WBC, white blood cells. (D) Flow cytometric analysis of bone marrow (BM) and spleen cells from MMTV-Cre; Cbl^flox/flox; Cblb^del/del and control (Cbl^flox/flox; Cblb^del/+) mice. Numbers indicate mean percentage ± SD of the Gr-1⁺, CD11b⁺ population (n = 3 each).

Upon necropsy, all MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice showed massive hepatosplenomegaly (Fig. S1A), moderately enlarged lymph nodes, and white marrow. Thymi were not enlarged in more than 20 animals submitted for detailed necropsy. Although mammary gland development was mildly delayed in female MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice, it was not immediately clear whether this was an intrinsic defect of Cbl, Cbl-b double deficiency within the mammary gland or secondary to poor health of the animals. Details of mammary phenotypes of these mice will be reported elsewhere.

Microscopic examination of tissue sections revealed signs of extramedullary hematopoiesis in the spleen with disruption of the normal splenic architecture (Fig. 1B). Livers had numerous foci composed of myeloid cells surrounding the sinusoids. In the bone marrow, marked myelopoiesis was observed. These observations were strongly suggestive of hematological disorders in mice. Therefore, we elected to further investigate the hematopoietic organs.

Analysis of the peripheral blood of MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice revealed prominent leukocytosis accompanied by an increase in the percentage and the absolute number of monocytes (Fig. 1C and Fig. S1B). Erythrocyte and platelet counts were within the normal range. Despite pronounced leukocytosis, no blasts were detected in the peripheral blood.

An increase in the myeloid/monocyte population was also confirmed by flow cytometry. As shown in Fig. 1D, there was a marked expansion of Gr1/CD11b-positive cells both in the bone marrow and in the spleen.

Altogether, these hematological/pathological findings are consistent with the diagnosis of MPD-like myeloid leukemia according to the Bethesda proposals (18).

MMTV-Cre Is Active in the Early Hematopoietic Compartment.

Although Cbl deletion in hematopoietic lineages was not the original intent of our studies, it has been reported that MMTV LTR promoter-driven gene expression is not limited to the mammary gland. In addition to the mammary epithelium, this MMTV-Cre allele was shown to function in the salivary gland, skin, and the hematopoietic/lymphoid system, among other tissues (17). We therefore hypothesized that the observed hematological phenotypes of the MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice were due to Cre expression in the hematopoietic/lymphoid system, and proceeded to examine this in detail.

We first determined whether the MMTV-Cre transgene used in our studies is active in the hematopoietic compartment. For this purpose, we crossed the MMTV-Cre mice to a Cre reporter strain, CAG-CAT-EGFP (19). In this reporter strain, GFP expression is detectable only after Cre-mediated excision of the loxP-flanked CAT gene located between the modified chicken actin promoter and the EGFP gene.

As expected, GFP was robustly expressed in the mammary gland of female MMTV-Cre; CAG-CAT-EGFP double transgenic mice (Fig. S2A). In the bone marrow, GFP was expressed in more than 10% of CD34⁻ LSK (Lin⁻Sca-1⁺c-Kit⁺) cells, demonstrating that the MMTV-Cre transgene is active in the early hematopoietic compartment, known to be highly enriched for long-term hematopoietic stem cells (LT-HSC) (Fig. S2B). However, the percentage of GFP-expressing cells in the more mature lymphoid and myeloid cells was less than 3%. This indicates that, in the absence of proliferative and/or survival advantages, cells that undergo Cre-mediated recombination make up a relatively small fraction of the overall hematopoietic/lymphoid compartment. However, when splenocytes from an MMTV-Cre; Cbl^flox/flox; Cblb^del/del mouse were analyzed, Cbl protein was undetectable by immunoblotting (Fig. 2A). Because Cblb is deleted in the germ line in the present breeding scheme, absence of Cbl indicates that the vast majority of cells in the spleen are deficient in both Cbl and Cbl-b. This finding was corroborated by immunohistochemical staining of bone marrow, spleen, and thymus of MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice, where anti-Cbl antibody staining was essentially undetectable (Fig. 2B).

Fig. 2.

CBL is absent in the hematopoietic/lymphoid compartment of MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice. (A) Immunoblot analysis of spleen tissue lysate. Spleen lysates were prepared from mice with the indicated genotypes and analyzed by immunoblotting using anti-Cbl antibody. The same blot was probed with anti-β actin antibody to ensure equal loading. (B) Immunohistochemical analysis of BM, spleen, and thymus. Paraffin sections were stained with anti-Cbl antibody and revealed by DAB, which shows brown staining. Control sample is of Cbl^flox/flox; Cblb^del/+ genotype. (Scale bars, 100 μm.)

These results clearly demonstrate that hematopoietic/lymphoid cells from MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice are doubly deficient in both Cbl and Cbl-b; we will therefore refer to the MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice as “double knockout” (dKO) in the remainder of this manuscript.

Lack of Cbl/Cbl-b Expands the Hematopoietic Stem/Progenitor Population in the Bone Marrow and Confers Hypersensitivity to Cytokines.

To understand the role of Cbl-family proteins in hematopoietic cell development in more detail, we first compared the cell-surface marker expression of bone marrow cells from dKO and control mice. Total bone marrow cellularity was comparable between dKO and control mice. However, the percentages as well as the absolute numbers of Lin⁻Sca-1⁺c-Kit⁺ (LSK) and Lin⁻Sca-1⁻c-Kit⁺ (LK) populations were significantly increased (Fig. 3A).

Fig. 3.

Myeloid stem/progenitor cell number is increased and bone marrow cells are hypersensitive to cytokines in MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice. (A) Flow cytometric analysis of bone marrow Lin⁻ population and absolute numbers of LSK (Lin⁻Sca-1⁺c-Kit⁺) and LK (Lin⁻Sca-1⁺c-Kit⁻) cells. Cell numbers were determined from two femurs and two tibiae and mean ± SD are shown in the bar graphs (n = 3). Differences in cell numbers between control and dKO samples were statistically significant by unpaired, two-tailed t test (P = 0.0018 for LSK, P = 0.0086 for LK). (B) Methylcellulose colony formation in response to cytokines. Unfractionated bone marrow cells (2 × 10⁴) were plated in triplicate plates and colonies were counted on day 7. The result shown here is representative of three independent experiments with similar results. Control samples (open bars) are of Cbl^flox/flox; Cblb^del/+ or Cbl^+/+; Cblb^+/+ genotypes. The asterisks indicate that no colonies with >50 cells were observed.

One of the characteristic features of MPD is increased sensitivity to cytokines (20). Therefore, we compared the responses of bone marrow cells to cytokines using the methylcellulose colony-formation assay. To simulate the cytokine sensitivity assays described for human JMML (21), we used unfractionated total bone marrow cells. As shown in Fig. 3B, dKO bone marrow cells formed more colonies than control cells when grown in the presence of various concentrations of cytokines. Furthermore, a small number of colonies formed reproducibly even in the absence of exogenously added cytokines in dKO cultures but not in control cultures.

Altered Response to Thrombopoietin.

Increased colony-forming abilities of dKO bone marrow cells may be due to increased frequency of cytokine-responsive cells or because individual dKO cells were more sensitive to cytokine stimulation. One potential approach to distinguish between these mechanisms is to compare the expression of cytokine receptors on various bone marrow cell subpopulations. However, this approach is limited by the lack of availability of antibodies suitable for flow cytometry and also by the heteromultimeric structure of most cytokine receptors. Expression of individual cytokine receptor subunits may not correlate with the levels of the signal-competent cytokine receptors.

Therefore, we chose to compare biochemical responses of bone marrow cells to cytokines instead. Flow cytometric analysis of intracellular signal transduction molecules has emerged as a powerful assay to assess biochemical responses to exogenous stimuli among rare cells that are not amenable to conventional immunoblot-based approaches (22). To evaluate the responses of hematopoietic stem/progenitor cells, we focused on c-Kit⁺ cells within the Lin⁻ population; this population contains both LSK and LK populations. We chose this approach, as we could not detect Sca-1 by flow cytometry after cell fixation and permeabilization. On the other hand, c-Kit staining remained robust after such manipulations. As shown in Fig. 4, both dKO and control bone marrow cells showed comparable responses to GM-CSF and stem cell factor (SCF) when measured using the intracellular levels of phosphorylated Erk (pErk) and phosphorylated STAT5 (pSTAT5). However, response to thrombopoietin (TPO) was altered in dKO cells; whereas the response to TPO peaked at 5 min after stimulation in control cells and diminished subsequently, the response continued to rise until 15 min of stimulation in dKO cells. These differences were observed reproducibly in multiple independent experiments.

Fig. 4.

Response to TPO is altered in MMTV-Cre; Cbl^flox/flox; Cblb^del/del bone marrow progenitors. Lin⁻ bone marrow cells were stimulated with GM-CSF (10 ng/mL), SCF (100 ng/mL), or TPO (100 ng/mL) as indicated, fixed, permeabilized, and stained with antibodies against c-Kit and anti-phospho-Erk or anti-phospho-STAT5. At least 100,000 events were collected for each sample. Data are expressed as the fold change of the mean fluorescence intensity (MFI) of pErk or pSTAT5 staining on the c-Kit⁺ population against MFI values without stimulation (time = 0). The results shown here are means ± SD of at least three independent experiments. Control samples are of Cbl^flox/flox; Cblb^del/+ or Cbl^+/+; Cblb^+/+ genotypes.

Transfer of MPD to Myelosuppressed NSG Mice.

The data presented above are all consistent with the idea that loss of Cbl plus Cbl-b in the hematopoietic stem/progenitor compartment leads to MPD in dKO mice. However, because Cbl-b is deleted in the germ line and also because MMTV-Cre functions in a wide range of epithelial and nonepithelial tissues, we sought to determine whether MPD phenotype is cell-autonomous or dependent upon a complex physiological milieu unique to this mouse model.

For this purpose, we transferred bone marrow cells from control and dKO mice into myelosuppressed NSG (NOD scid γ) mice (23). Because leukocytes derived from NSG and donor mice differ in their CD45 allele (recipients express CD45.1, donors express CD45.2), cell origins can be readily distinguished by cell-surface staining. Out of five mice assigned to each experimental group, one recipient of dKO bone marrow died during bone marrow cell injection due to technical complications and another died 50 d after transfer; one control bone marrow recipient died 52 d after transfer. Peripheral leukocyte counts could not be determined for these animals. All of the surviving animals (four control and three dKO bone marrow recipients) were bled 8 wk after bone marrow transfer. As shown in Fig. S3A, dKO recipients showed a significant increase in donor-derived cells, a hallmark of MPD. Spleens and livers of dKO bone marrow recipients showed myeloid infiltration similar to that seen in MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice (Fig. S3B).

Discussion

Although the Cbl-family E3 ubiquitin ligases are now widely viewed as critical components of negative regulation of signaling downstream of tyrosine kinase-coupled cell-surface receptors, how these proteins control tissue homeostasis is much less clear. The need to understand the functional importance of this evolutionarily conserved protein family is highlighted by the linkage of Cbl protein to regulation of HSC pool size (14) and recent identification of MPD-associated mutations in CBL (4–13). Here we report that Cbl and Cbl-b, closely related members of the mammalian Cbl gene family, function redundantly to limit the size of the HSC pool and that combined deletion of both Cbl and Cbl-b produces a rapidly lethal MPD.

Our data demonstrate that somatic loss of Cbl on a germ-line Cbl-b-deficient background leads to lethal early-onset MPD. MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice show marked myeloid expansion both in the bone marrow and in the periphery, and this phenotype was fully penetrant. Importantly, bone marrow cells can transfer MPD to myelosuppressed hosts, demonstrating the cell-autonomous nature of this phenotype. Development of a rapidly progressive MPD in this model is particularly remarkable in view of previous studies that Cblb-null background confers resistance to both transplanted and spontaneously arising tumors (24, 25). Clearly, the Cbl-b-deficient genetic background did not interfere with the pathogenesis and progression of MPD in the current model. Using a GFP reporter strain, we demonstrated that, in addition to its anticipated activity in the mammary gland, the MMTV-Cre allele was active in the hematopoietic cell lineages. Despite the aggressive disease phenotype, however, the MMTV-Cre activity in the bone marrow is surprisingly modest: Only 10% of CD34⁻ LSK cells (HSCs) and less than 3% of total bone marrow cells expressed GFP when crossed to a reporter strain (Fig. S2B).

The present findings have significant implications for the role of Cbl proteins in HSC regulation and for MDS/MPDs associated with CBL mutations. It is notable that the level of HSC expansion in our model is only modestly more than that reported in Cbl-null mice (14), yet there is no evidence of an early-onset lethal MPD in Cbl-null mice. This difference is most likely due to the presence of Cbl-b in Cbl-null mice, pointing to the redundant yet critical role of Cbl-b in the HSC proliferation/differentiation program. Beyond the simpler and testable explanations such as up-regulation of Cbl-b expression at some critical stage in HSCs, it is conceivable that Cbl-b might have a relatively selective role in HSCs that has not been reported thus far. Obviously, lack of a hematopoietic developmental defect in Cblb-null and Cbl flox/Cblb-null mice clearly indicates that loss of Cbl-b function alone is insufficient to alter the HSC program. Studies in model cell systems have suggested biochemical differences between Cbl and Cbl-b (26). Our mouse model should allow future reconstitution studies to dissect out redundant versus specific roles of Cbl and Cbl-b in HSC regulation.

Interestingly, whereas Cbl (14) or Cbl/Cbl-b deficiency expands the earliest identifiable HSC population, the progeny of these cells is dramatically enriched for myelomonocytic lineage cells. Preferential expansion of the myelomonocytic cells is concordant with clinical manifestations in human patients with CBL mutations (7–11). Whether the skewing might reflect a Cbl/Cbl-b-dependent restriction point that regulates differentiation of HSCs toward myelomonocytic lineage or regulation of committed myelomonocytic precursors are important questions that should be possible to address using the current model.

In this regard, it is notable that Cbl is among a minority of HSC regulators whose loss leads to expansion of HSCs without exhausting their long-term hematopoietic reconstitution potential (27). The ability of the Cbl/Cbl-b dKO bone marrow to transfer MPD to recipient animals suggests that Cbl proteins are unlikely to function in the bone marrow niche but rather directly on HSCs to regulate the HSC pool size or myelomonocytic differentiation. One potential mechanism of interest in this regard is the hypersensitivity of dKO HSCs toward cytokine TPO (Figs. 3 and 4).

The similarity of manifestations of a combined Cbl and Cbl-b deficiency in HSCs with the disease spectrum seen in CBL mutation-associated AML/MPD/JMML patients is of considerable significance despite the fact that human diseases are caused by missense mutations or small deletions clustered around the linker/RING finger domains. Given this similarity, and the lack of a comparable lethal MPD when Cbl alone is deleted (28, 29), it is reasonable to surmise that mutant Cbl proteins found in human disease might not only function as dominant-negative inhibitors of Cbl but also of Cbl-b. Thus, the model system presented here should provide a clean background to test whether and how the expression of mutant Cbl proteins in the absence of wild-type Cbl/Cbl-b may alter downstream biochemical pathways and affect disease progression.

To better understand the mechanisms of mutant Cbl-associated MPD, attributes of currently available animal models should provide useful insights. Aside from reported null mutant models (11, 28, 29) which do not produce lethal MPD, four mouse models expressing mutant Cbl proteins have been reported. A knock-in mouse model to abrogate the interaction of the Cbl TKB domain with its targets (via G304E mutation) produced a mild myeloid phenotype without significant effects on T-cell development (30). On the other hand, C379A RING mutant knock-in mice were born at sub-Mendelian ratios and thymocyte numbers were significantly reduced (31). Interestingly, C379A mutant mice appear to develop MPD (13). A mutation in one of the key tyrosine residues in the C-terminal region (Y737F mutation) does not appear to lead to significant lymphoid/hematological phenotypes (32). Recently, Bandi and colleagues used retrovirus-mediated gene transfer to assess the consequences of introducing the 70Z Cbl (linker deletion) mutant and a leukemia patient-derived RING finger domain mutant (R420Q) of Cbl into bone marrow cells (33). In this model, mice developed hematological disorders with an average survival period of over 300 d. The authors reported that all animals expressing mutant Cbl developed diffuse mastocytosis in addition to MPD and myeloid leukemia with maturation.

In the MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice described here, the disease onset was early and, in more than 20 mice submitted to detailed pathological/hematological evaluation, no animals showed signs of mastocytosis or frank myeloid leukemia. One of the key differences between our model and those reported is the presence of endogenous Cbl/Cbl-b (in the Bandi et al. study) or Cbl-b (for the knock-in studies). Because mutant Cbl proteins are most likely to function, at least in part, by inhibiting the functions of normal Cbl/Cbl-b, the presence of endogenous wild-type Cbl proteins is likely to have influenced the outcome of expressing mutant Cbl proteins in previous studies. Also, gene expression levels upon retrovirus-mediated gene transfer depend heavily on the activity of the virus promoter and on the integration site of the virus genome that are also likely to be factors in the Bandi et al. study. Finally, the mouse strain background may also influence the disease phenotypes. Bandi and colleagues used BALB/c mice as bone marrow donors as well as recipients, whereas our MMTV-Cre; Cbl^flox/flox; Cblb^del/del mice are on a C57BL/6 background. Future studies with patient-derived mutants expressed from endogenous Cbl promoter should help resolve these discrepancies.

Although JMML is a relatively rare childhood malignancy, studies of its pathogenesis have yielded critical insights into the molecular mechanisms of MPD. Seventy to seventy-five percent of JMML cases arise from mutations in NRAS, KRAS, PTPN11, or NF1 (34). Recent reports estimate that CBL mutations account for a significant fraction of the remaining cases (10, 13). A recurring theme across these mutations is a deregulation of the Ras-Erk signaling pathway. Mouse models of these genetic lesions recapitulate many of the clinical features of JMML and have helped establish the causal roles of patient-derived mutations in the development of JMML. Yet, these mouse models have also revealed some distinct features of individual JMML-associated mutations.

Notably, similar to findings in JMML and several JMML-related mutant mouse models (reviewed in ref. 34), Cbl, Cbl-b dKO bone marrow cells were hypersensitive to GM-CSF and able to form colonies even in the absence of exogenous cytokines. However, whereas both Kras^G12D and Ptpn11^D61Y mutant mice show a reduction in the number of LSK cells in the bone marrow (35, 36), this population is expanded in the bone marrow of Cbl-deficient (14) and Cbl/Cbl-b double-deficient mice (Fig. 3). Likewise, despite enhanced methylcellulose colony formation by dKO bone marrow cells in response to various cytokines, we did not observe major alterations in early signaling events downstream of cytokine stimulations between Cbl/Cbl-b double-deficient Lin⁻c-Kit⁺ cells and controls. Unlike Kras^G12D or Ptpn11^D61Y mutants, receptor-downstream signaling pathways were not hyperactivated by GM-CSF or SCF in Cbl/Cbl-b double-deficient cells, and the only difference we observed was a small but reproducible prolongation of the response to TPO stimulation in dKO cells. These data are essentially in accord with findings with Cbl-deficient HSCs (14). Given the roles that TPO/TPO-R (c-Mpl) play in the regulation of HSCs (37, 38), expansion of the LSK population we observe, and the known ability of Cbl to interact with TPO-R (39), the enhanced activation of TPO-R may be directly related to deregulation of the HSC compartment in the present model. Taken together, these observations point to a potentially unique role of Cbl-family proteins in regulating HSCs and how their alterations might unleash a neoplastic program. Careful functional characterization of HSCs comparing the previously reported models with the one described here may therefore help elucidate key issues related to JMML and other MPD pathogenesis.

Methods

Mice.

The Cbl floxed, Cblb-null mouse strain B6.Cg-Cbl^tm2Hua Cblb^tm1Hua (Cbl^flox; Cblb^del) and the Cre reporter transgenic mouse strain CAG-CAT-EGFP were reported previously (15, 19). The transgenic mouse strain with a single copy of Cre recombinase driven by the MMTV promoter, STOCK Tg(MMTV-cre)4Mam/J [corresponds to the D line in the original report (17)] and the NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NOD scid γ; NSG) mice used for hematopoietic repopulation assays were purchased from The Jackson Laboratory. Cbl^flox; Cblb^del and MMTV-Cre transgenic mice were backcrossed to C57BL/6 for at least five generations before being intercrossed to generate MMTV-Cre; Cbl^flox; Cblb^del mice. All animals were housed under specific pathogen-free conditions in the animal care facility at the Center for Comparative Medicine of the University of Nebraska Medical Center (UNMC). All mouse experiments were approved by the Institutional Animal Care and Use Committee of the UNMC. Mice were analyzed between 4 and 8 wk of age unless otherwise specified.

Reagents and Antibodies.

Citric acid-based antigen unmasking solution, M.O.M. immunodetection kit, VECTASTAIN ABC kit, and 3,3′-diaminobenzidine (DAB) substrate kit were from Vector Laboratories. Cytokines were from PeproTech. MethoCult M3234 methylcellulose medium was from Stem Cell Technologies. Busulfan and Cremophor EL were from Sigma-Aldrich. Anti-Cbl monoclonal antibody (clone 17) was from BD Biosciences. The following antibodies for flow cytometry were either from BD Biosciences or from eBioscience: Ly-6G (Gr-1, RB6-8C5); CD11b (Mac-1, M1/70); CD45R (B220, RA3-6B2); CD45.1 (A20); CD45.2 (104); Ly-6A/E (Sca-1, D7, or E13-161.7); CD117 (c-Kit, 2B8); CD34 (RAM34). Anti-phospho-STAT5 monoclonal antibody (clone 47) was from BD Biosciences. Anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (D13.14.4E) was from Cell Signaling Technology.

Pathology.

Tissues were fixed in Fekete's acid alcohol formalin overnight at room temperature, transferred to 70% ethanol, embedded in paraffin, and sectioned. Bones were decalcified in 10% EDTA solution before embedding. For immunohistochemistry, sections were deparaffinized, rehydrated, and boiled in antigen unmasking solution. After blocking endogenous peroxidase activity, the sections were blocked with M.O.M. mouse Ig blocking reagent, incubated with anti-Cbl monoclonal antibody at 1:500 followed by incubation with M.O.M. biotinylated anti-mouse Ig, and revealed by VECTASTAIN ABC kit using DAB as a substrate. Slides were counterstained with hematoxylin. Complete blood counts and white blood cell differential analysis were performed at IDEXX.

Flow Cytometry.

Single-cell suspensions were prepared from bone marrow (femurs and tibiae) and spleen. Spleen samples were treated with ACK lysis buffer (150 mM NH4Cl, 10 mK KHCO3, 0.1 mM Na2EDTA) to remove erythrocytes. The cells were stained with antibodies as indicated in the figures. Data acquisition was performed on a FACSCalibur or LSRII at the UNMC Cell Analysis Facility and data were analyzed using FlowJo software (Tree Star).

For HSC/progenitor analysis, cells expressing lineage markers (Lin⁺) were labeled with biotin-conjugated antibodies against CD5, B220, CD11b, Gr-1, and 7-4 (mouse lineage depletion kit; Miltenyi Biotec) and magnetically depleted using autoMACS (Miltenyi Biotec) before surface staining.

Lineage marker-depleted bone marrow cells were used for intracellular phosphoprotein analysis. Cells were prepared essentially as described previously with minor modifications (40). After autoMACS, cells were left untreated or stimulated with cytokines as indicated and the stimulation was stopped by adding paraformaldehyde solution directly to the mixture to a final concentration of 1.6%. After permeabilizing with acetone, the cells were stained with the indicated antibodies.

Methylcellulose Colony Assay.

Unfractionated bone marrow cells were mixed with MethoCult M3234 methylcellulose medium (Stem Cell Technologies) at 2 × 10⁴/mL in the presence of the indicated amounts of cytokines and plated in 1-mL triplicate cultures. Colonies were counted on day 6 or 7. Only cell clusters with more than 50 cells were counted as colonies.

Adoptive Transfer of Bone Marrow Cells.

Busulfan was dissolved in DMSO at 100 mg/mL and mixed with nine volumes of 50% Cremophor formulation (10% Cremophor EL, 15% propylene glycol, 25% ethanol, 50% PBS). Immediately before injection, the above solution was diluted with 5% glucose to adjust the final concentration of busulfan to 4 mg/mL. NSG mice were conditioned by injecting two doses of 20 mg/kg body weight busulfan intraperitoneally 24 h apart (23). Twenty-four hours after the second busulfan injection, 2 × 10⁶ total bone marrow cells were injected intravenously.