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Cancer Research 67, 7565, August 15, 2007. doi: 10.1158/0008-5472.CAN-07-1467
© 2007 American Association for Cancer Research
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Priority Reports

Myeloproliferative Defects following Targeting of the Drf1 Gene Encoding the Mammalian Diaphanous–Related Formin mDia1

Jun Peng1, Susan M. Kitchen1, Richard A. West1,2, Robert Sigler3, Kathryn M. Eisenmann1 and Arthur S. Alberts1

1 Laboratory of Cell Structure and Signal Integration and 2 Flow Cytometry Core Facility, Van Andel Research Institute, Grand Rapids, Michigan and 3 Esperion Therapeutics, Division of Pfizer, Ann Arbor, Michigan

Requests for reprints: Art Alberts, Research Laboratories, Van Andel Research Institute, 333 Bostwick Avenue N.E., Grand Rapids, MI 49503. Phone: 616-234-5316; Fax: 616-234-5317; E-mail: Art.Alberts{at}vai.org.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rho GTPase-effector mammalian diaphanous (mDia)–related formins assemble nonbranched actin filaments as part of cellular processes, including cell division, filopodia assembly, and intracellular trafficking. Whereas recent efforts have led to thorough characterization of formins in cytoskeletal remodeling and actin assembly in vitro, little is known about the role of mDia proteins in vivo. To fill this knowledge gap, the Drf1 gene, which encodes the canonical formin mDia1, was targeted by homologous recombination. Upon birth, Drf1+/– and Drf1–/– mice were developmentally and morphologically indistinguishable from their wild-type littermates. However, both Drf1+/– and Drf1–/– developed age-dependent myeloproliferative defects. The phenotype included splenomegaly, fibrotic and hypercellular bone marrow, extramedullary hematopoiesis in both spleen and liver, and the presence of immature myeloid progenitor cells with high nucleus-to-cytoplasm ratios. Analysis of cell surface markers showed an age-dependent increase in the percentage of CD11b+-activated and CD14+-activated monocytes/macrophages in both spleen and bone marrow in Drf1+/– and Drf1–/– animals. Analysis of the erythroid compartment showed a significant increase in the proportion of splenic cells in S phase and an expansion of erythroid precursors (TER-119+ and CD71+) in Drf1-targeted mice. Overall, knocking out mDia1 expression in mice leads to a phenotype similar to human myeloproliferative syndrome (MPS) and myelodysplastic syndromes (MDS). These observations suggest that defective DRF1 expression or mDia1 function may contribute to myeloid malignancies and point to mDia1 as an attractive therapeutic target in MDS and MPS. [Cancer Res 2007;67(16):7565–71]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Formins nucleate, processively elongate, and (in some cases) bundle filamentous actin (F-actin) through conserved formin homology-2 domains (1). The mammalian diaphanous (mDia)–related formins participate in many cytoskeletal remodeling events, including cytokinesis, vesicle trafficking, and filopodia assembly, while acting as effectors for Rho family small GTPases (2). Active GTP-bound Rho binds to and activates mDia proteins through disruption of an intramolecular mechanism maintained by conserved regulatory domains that flank the formin homology-2 domain (3, 4).

To date, there are only two genetic disorders associated with the genes encoding mDia proteins (2). In the first, the DFNA1 allele of the DRF1/DIAPH1 (5q31) gene for human mDia1 has been characterized in nonsyndromic deafness (5). The DFNA1 mutation is likely not a loss-of-function mutation, however. The allele carries a frameshift mutation predicted to cause a truncation near the C-terminal diaphanous autoregulatory domain (4) that may affect mDia1 autoinhibition and regulation by small GTPases (6). The second DRF genetic defect is a breakpoint translocation in the last exon of the DRF2/DIAPH2 (Xq22) gene encoding human mDia3 protein; the translocation has been associated with premature ovarian failure in one patient (7). However, there has been no demonstration that expression or function of mDia3 protein was affected by this mutation.

In this study of the effects of knocking out Drf1 gene expression in mice, we show that mDia1 plays an essential role in myelopoiesis. As animals age, they develop myeloproliferative defects in both the bone marrow and peripheral blood. These observations point to a crucial role of mDia1 in maintaining myeloid homeostasis, potentially by functioning as a tumor suppressor or susceptibility gene.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gene targeting, immunoblotting, and genotyping. Gene targeting, genotyping, and immunoblotting were done exactly as described in Peng et al. (8). Embryonic stem cells used in the initial targeting were from 129(CJ7) and chimeric mice were B6; mice used in this study were of a mixed 129/B6 background.

Fluorescence-activated cell sorting analysis. Marrow, spleen, blood, and tumor cells (where applicable) from each age group and genotype were characterized by flow cytometric analyses. Marrow was flushed from femurs using a syringe with a fine gauge needle and 3 mL of PBS. Single-cell suspensions of spleen and tumors were obtained by mincing tissue with glass slides and subsequent passage and scraping of tissue in a ThermoShandon biopsy bag (Thermo Fisher Scientific). Cells were incubated for 15 min. at 20°C in the dark. Incubation was followed by addition of 1x FACSLyse reagent (Becton Dickinson) for 15 min at 20°C in the dark. After RBC lysis the remaining cells were washed in 2 mL PBS with 0.1% sodium azide. Cells were fixed in 1.0% methanol-free formaldehyde (Polysciences, Inc.) in PBS containing 0.1% bovine serum albumin and refrigerated at 4.0°C until acquisition. Appropriate subclass and negative controls were used to detect nonspecific binding of antibody and autofluorescence. A minimum of 10,000 events for fresh marrow mononuclear cells and 5,000 events for spleen and tumor cells were acquired when possible. Flow cytometric analyses were conducted using either a Becton Dickinson FACSCalibur four-color or a FACSAria 12-color flow cytometer (Becton Dickinson). Data were analyzed using Becton Dickinson CellQUEST Pro® (v5.2.1) and FACSDiVa® (v5.0.1) software.

Monoclonal antibodies. The following monoclonal antibodies were used: anti-CD14FITC (Sa2-8) from eBioscience; anti-CD29APC from BioLegend; anti-CD45PerCP (30-F11), anti-CD41FITC (MWReg30), anti-CD71FITC (C2), anti-CD74FITC (In-1), anti-TER-119PE (Ly-76, TER-119), anti-CD13PE (R3-242), anti-CD19PE (1D3), and anti-CD11bAPC (D12) from BD PharMingen; anti-CD8aPE (5H10), anti-CD4APC (RM4-5), anti-CD34APC (MEC14.7), and anti-CD3FITC (500A2) from Invitrogen/Caltag Laboratories.

Cell cycle analysis. Cell cycle analyses were done where applicable using propidium iodide (Sigma) in a modified Vindelov's preparation. Whenever possible, a minimum of 10,000 events were collected by flow cytometry. Data were analyzed using Becton Dickinson CellQUEST Pro and Verity House ModFIT LT (v3.1) software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the murine Drf1 gene encoding the mammalian diaphanous-related formin mDia1 (9) protein was disrupted by homologous recombination in embryonic stem cells derived from 129Sv mice as previously described (8, 10); the targeting strategy is shown schematically in Fig. 1A . Drf1-targeted embryonic stem cells were used to generate 129Sv/B6 chimeras and the Drf1 mice were maintained on a B6/129Sv background. Loss of mDia1 protein expression in Drf1+/– and Drf1–/– mice was confirmed by immunoblotting (Fig. 1A). At birth, mDia1 Drf1–/– animals were viable and morphologically normal.


Figure 1
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  		Figure 1. Drf1 gene targeting leads to dermatoses, splenomegaly, and myelodysplasia. A, gene targeting strategy as reported previously along with example PCR-based genotyping (8). Immunoblotting for mDia1 expression in 20 µg of whole-cell lysate using P155 rabbit anti-mDia1 (20). B, Drf1–/– mice (male and female, inset; 450 d) with malocclusion (top), granulated dermatitis, and inflammation on paws. Female with dermatoses along crest and alopecia of muzzle from excessive barbering (bottom). H&E staining of formalin-fixed paraffin-embedded skin section with chronic inflammation (without any evidence of infection) and high levels of neutrophil invasion (inset). C, splenomegaly in Drf1-targeted mice (scale = cm). The histogram shows mean wet weight of spleens at necrocropsy of age-matched (450 d) mice. Columns, mean; bars, +/– standard deviation. D, H&E-stained splenic sections showing a lack of splenic nodules and T-cell infiltration sections from 450-day-old mice of the indicated genotypes.

 
As animals aged (>300 days of age), however, they frequently (30–40%) developed dermatoses and, in some cases, alopecia (Fig. 1B). H&E staining of formalin-fixed and paraffin-embedded skin sections routinely showed no sign of infection but high levels of neutrophil invasion (inset). In addition to these outward signs of ill health, animals often failed to thrive between 200 and 450 days of age and showed signs of dehydration and anemia (pale ears and feet). Despite these defects, life spans of Drf1+/– and Drf1–/– mice were not significantly different than those of their wild-type littermates (data not shown).

Upon necropsy of animals found dead or euthanized at various ages, splenomegaly (Fig. 1C) was observed in both Drf1+/– and Drf1–/– mice; however, the difference in spleen mass (450 days of age) was statistically significant only in Drf1–/– animals (Fig. 1C, histogram). Histopathology revealed significant dysplasia in the spleen, as shown in H&E-stained sections (Fig. 1D) from 100-day-old Drf1-null mice. Typically, sections showed a lack of or malformed germinal centers with essentially no white pulp within the spleen, suggesting a defect in immune cell migration and/or proliferation. Similar observations were made in lymph nodes of Drf1-targeted mice (data not shown).

Consistent with the observations in 100-day-old Drf1–/– animals, splenic sections from older Drf1+/– animals (~400 days) had poorly formed germinal centers but also had increased levels of granulocytes and neutrophils (Fig. 2A ). Additional features included high levels of hemosidirin (a breakdown product of hemoglobin), marked congestion and necrosis, and extramedullary hematopoiesis (EMH). EMH was also observed in the lung and liver (data not shown; Fig. 2A, far right), where it was accompanied by aggregates of plasma cells.


Figure 2
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  		Figure 2. Myelodysplastic features in spleen and bone marrow. A, dysplasia in formalin-fixed H&E-stained paraffin-embedded splenic sections in 300-day-old to 450-day-old mice with indicated genotypes (described in detail in text). B, bone marrow smears from femurs of 400-day-old mice stained with Wright-Giemsa. C, Wright-Giemsa–stained peripheral blood from 400-day-old littermates.

 
Bone marrow was examined to assess what, if any, changes might have occurred in the myeloid compartment. Figure 2B shows Giemsa-Wright–stained bone marrow obtained from ~400-day-old mouse femurs. Whereas Drf1+/+ mice displayed normal marrow, marrow from both Drf1+/– and Drf1–/– animals were markedly hypercellular. Erythroid progenitor cells contained abnormal mitotic figures, abnormally high nucleus-to-cytoplasm ratios, and increased levels of monocytes and ring granulocytes. In rare cases, the marrow was fibrotic, as shown in the bottom-right of Fig. 2B (>5% of examined smears). Peripheral blood characteristics of Drf1-targeted mice were then examined.

Blood smears of age-matched 450-day-old mice showed varying degrees of dysplasia; representative examples are shown in Fig. 2C compared with blood from wild-type mice. Peripheral blood smears from Drf1+/+ and Drf1+/– were indistiguishable. However, peripheral blood from age-matched Drf1–/– littermates was abnormal by comparison. An example is shown in Fig. 2C (third column and inset) where abnormally shaped (dysplastic) erythrocytes, characterized as echinocytes with spiked or star-shaped appearance, were seen in Drf1–/– samples; hemoglobin levels were within normal ranges (data not shown). The same animal had numerous immature myeloid progenitors. This was also observed in a littermate as shown in Fig. 2C (4th column); echinocytes were not present in those samples. In both cases, the progenitor cells seemed clumped with high nucleus to cytoplasm ratio. The WBC count in peripheral blood was abnormally elevated in this particular mouse (51.3 x 103/µL blood) and was significantly elevated in a cohort of 10 Drf1–/– mice compared with Drf1+/– and Drf1+/+ age-matched littermates (Supplementary Table S1). The high WBC levels and other hyperproliferative features in myeloid progenitors prompted us to examine myelopoiesis in more detail.

We used flow cytometric analysis to segregate bone marrow cells from 100-day-old and 450-day-old mice (n > 15, each genotype). By plotting CD45 (a pan-leukocyte marker) versus side-scatter, cells segregated into discrete populations: blasts (immature cells), erythroid precursors (EPC), lymphocytes (lymph), monocytes (monos), and granulocytes (grans; as shown in the gating strategy outlined in Fig. 3A ). We found that 450-day-old Drf1+/– and Drf1–/– mice had significant expansion of cells in the granulocyte compartment (Fig. 3B). In contrast, the percentage of cells falling into the lymph gate was diminished; these results were consistent with the lymphopenia documented in the Supplementary Table S1. Cells within the respective gates were then analyzed further to determine the developmental status of cells within each compartment.


Figure 3
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  		Figure 3. Myeloid bone marrow composition and myeloid components of marrow. A, bone marrow composition for Drf+/+, Drf1+/–, and Drf1–/– 450-day-old mice. Symbols above the columns in parts B and C (*, bullet, °, {infty}, +, **, °°, and {emptyset}) denote the paired comparisons having significant P values. P values for the comparisons are noted below. B, bone marrow shows significant differences in composition at 450 d (P < 0.01; except for bullet, where P < 0.05). C, comparison of the CD14+ cells from marrow and spleen at 100 and 450+ days (P < 0.01; except for bullet and °, where P < 0.05). There was a significant increase in CD11b+ cells in marrow; in the spleen there were significant differences in each genotype at each time point (P < 0.05; except for °, where P < 0.01). CD29 expression differences were found in marrow only in the Drf1-null relative to the other genotypes at 450+ days (P < 0.01). In spleen, there are significant differences seen at 100 d only in the null compared with the other genotypes (P < 0.05); at 450+ days there were significant differences seen in all three genotypes (P < 0.01; except for °, where P < 0.05). In both B and C, bars represent the means +/– standard deviations.

 
CD14 (LPS/LBP receptor; an activation marker of monocyte and macrophage cell lineage) expression vs. CD11b (integrin {alpha}M; monocyte development marker) was assessed in spleens and bone marrow from mice, ages 100 and 450 days (Fig. 3C). Consistent with the observations shown in Fig. 3B, the percentage of cells expressing the monocyte marker CD11b remained relatively unchanged within the bone marrow across all genotypes. As animals aged, however, a 2-fold increase in the percentage of CD11b+ cells was observed in spleens of Drf1–/– animals compared with wild-type animals. The percentages of CD14+ cells within either bone marrow or spleen was enhanced in both Drf1+/– and Drf1–/– mice relative to wild-type counterparts. The percentages of CD14+ cells within both marrow and spleen were also increased in a time-dependent manner in Drf1–/– mice. Collectively, these data are consistent with the notion that the activation status of the monocyte/macrophage population is enhanced upon loss of mDia1 protein expression.

We next examined the percentages of bone marrow and splenic cells—again in the lymph/mono gate—expressing the extracellular matrix receptor ß1 integrin (CD29), as its expression is important for both homing to and retention within lymphoid organs (Fig. 3C, bottom two histograms). The percentage of cells expressing CD29 in marrow was not altered in either Drf1+/– or Drf1–/– genotype. In contrast, the percentage of gated splenic cells expressing CD29 was significantly increased in both Drf1+/– and Drf1–/– mice, when compared with wild-type littermates. This increase within the spleen was amplified as the Drf1-targeted mice aged. This observation points to defects in the development and differentiation within the myeloid compartment of Drf1–/– mice potentially driving the development of splenomegaly. To further explore this possibility, erythropoiesis was analyzed in Drf1-targeted mice.

Figure 4 illustrates a comparison of TER-119 (erythroid-specific marker) and CD71 (transferrin receptor; marker of proliferating erythroid precursors) levels in bone marrow and spleen cells from 100-day-old and 450-day-old Drf1+/– and Drf1–/– animals relative to their wild-type counterparts. No significant differences were observed in either the relative percentages of TER-119+–gated or CD71+-gated splenic cells in 100-day-old mice (data not shown). Yet, by 450 days of age, there was a marked elevation in the percentage of gated Drf1–/– splenic cells expressing both CD71 and TER-119 (Fig. 4A). These data, particularly the high levels of transferrin receptor, led us to examine whether an increase in proliferation of erythroid progenitors within the spleen accounted for the observed EMH.


Figure 4
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  		Figure 4. Erythroid extramedullary and myeloid dysplasias. A, erythroid precursor compartment in both spleen and marrow at 450 d, where there is an increase in the number of erythroid precursors in the spleen in the Drf1-null (as in the extreme example in this figure, where 70% of the splenic tissue is erythroid precursor cells). See Fig. 3 caption for meaning of the symbols above the bars in B. B, erythroid precursors in spleen and marrow shows that the condition affects the Drf1-null animal in splenic tissue (P < 0.01). Also at 100 and 450 d, there is an increase in the splenic S phase (P < 0.01) but not in the marrow.

 
To test this hypothesis, the proportion of cells in S phase within bone marrow and the spleen were analyzed, as described in the Materials and Methods. As shown in Fig. 4B (right), the proportion of cells in S phase between different genotypes in cells from bone marrow was unchanged at both 100 or 450 days. Consistent with our hypothesis, at 450 days, there was a 4-fold to 6-fold increase in S phase in Drf1–/– animals and a 2-fold to 4-fold increase in the splenic erythroid precursors from Drf1+/– mice. These data, in combination with the TER119 and CD71 results, suggest that the rapidly proliferating cells within the spleen were erythroid precursors.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Knocking out mDia1 expression interferes with several aspects of myelopoiesis and erythropoiesis, with the severity of certain phenotypic aspects progressing with age. The complex Drf1–/– phenotype includes the potential to develop neutrophilic dermatoses, splenomegaly, EMH, alopecia, and numerous cytopenias and cytoses (11). Whereas the variability seen here may reflect the mixed genetic background of the mice, overall, the phenotype resembles human chronic myeloproliferative syndromes (MPS) and myelodysplastic syndromes (MDS). Both MPS and MDS have been characterized as preleukemic states, including variable lymphopenia, excess or dysfunctional erythrocytes, chronic myelomonocytic leukemia, ineffective hematopoiesis, and, in some cases, advancing myelofibrosis. There are also incidences of neutrophilic dermatoses (Sweet's syndrome) which can accompany MDS and MPS (12). MDS is a frequent hematologic disorder that affects typically older patients and is thought to be a stem cell disorder. Dysplastic features of the nucleus or cytoplasm, as observed in the mDia1 KO mice, and altered cellularity of the bone marrow are also characteristic of MDS (11).

mDia1 knockout leads to key hyperproliferation within the spleen and expansion of activated monocytes/macrophages within bone marrow and spleen. Consistent with MPS and MDS, active cell turnover and cell division was observed. MDS and MPS give rise to clonal hematopoietic and myelopoietic defects, and whereas we have not shown clonality, we consistently see defects in erythropoiesis. The effect of Drf1 gene targeting and the resulting mDia1 knockout suggests that the DRF1 gene for human mDia1 is affected in MPS, MDS, or other preleukemic pathologies.

DRF1 has been mapped to 5q31.3. There are several candidate genes in chromosome 5q in which defective expression/function could lead to myeloproliferitive defects and myelodysplasia (11). One is the gene encoding nucleophosmin NPM1, which has been targeted in mice (13). Whereas Npm1 haploinsufficiency led to some myeloproliferative defects in mice similar to those observed here, the NPM1 (5q35) gene often falls outside of the chromosomal abnormalities that accompany MDS (14). Another study has pointed to repressed {alpha}-catenin (CTNNA1; 5q31.2) gene expression in 5q– (minus) syndrome (15); CTNNA1 has been shown to fall within a minimal region of 5q31 deleted in some forms of acute myelogenous leukemia (AML)–associated with MDS (16). Whereas CTNNA1 message is repressed, the loss of or diminished {alpha}-catenin protein expression in primary patient samples has yet to be shown.

Recent evidence from carcinogen-treated mice also points to defects in Egr1/Krox-20 (5q31.2) expression as having a role in MDS and progression to AML (17). This finding is particularly compelling because expression of Egr-1 (an immediate-early gene product) is controlled by the serum response factor (SRF; ref. 18). Whereas Egr-1 expression is not entirely dependent upon Rho GTPase activity, its expression requires SRF activation via controlled actin dynamics (19). SRF in turn can be strongly induced by activated variants of mDia1, which potently assemble F-actin in cells (20). Therefore, loss of mDia1 may affect Egr-1 expression, thereby leading to similar outcomes as those observed in Egr-1–targeted mice.

Taken as a whole, the wide range of observations suggest that there are multiple mechanisms that lead to progression to myeloproliferative disease and progression to malignacy. The addition of the Drf1-targeted mice to this repertoire should help elucidate the molecular pathogenesis of myeloproliferative and myelodysplastic disease.


   Acknowledgments
 
Grant support: Van Andel Foundation, National Cancer Institute grant CA107529, American Cancer Society grant RSG-05-033-01-CSM (A.S. Alberts, J. Peng, and S.M. Kitchen), and NRSA grant F32 GM723313 (K.M. Eisenmann).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank David Nadziejka and Aaron DeWard for critical reading of the manuscript, Nick Duesbery for many helpful discussions, Bart Williams for critical insight, and members of Miranti, MacKiegan, and Duesbery laboratories for comments throughout the project.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/20/07. Revised 6/ 7/07. Accepted 6/21/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online