Transgenic Mice Expressing the P75 CCAAT-Displacement Protein/Cut

Research Article

Transgenic Mice Expressing the p75 CCAAT-Displacement Protein/Cut Homeobox Isoform Develop a Myeloproliferative Disease–Like Myeloid Leukemia Chantal Cadieux,1,2 Sylvie Fournier,5 Alan C. Peterson,1,3,4,6 Christian Be´dard,9,10 Barry J. Bedell,7,8 and Alain Nepveu1,2,3,4

1Molecular Oncology Group, McGill University Health Center, Departments of 2Biochemistry, 3Oncology, 4Medicine, 5Microbiology and Immunology, 6Human Genetics, and 7Pathology, McGill University; 8McConnell Brain Imaging Centre, Montreal Neurological Institute; andDepartments of 9Pathology and 10Microbiology, Faculty of Veterinary Medicine, Universite´de Montre´al, Montreal, Quebec, Canada

Abstract (Cux1) andCut-homeobox 2 ( Cux2) in mouse andchicken (3–5). The p75 CCAAT-displacement protein/Cut homeobox (CDP/ Hereafter in the text, the term CDP/Cux will be usedto describe Cux) isoform was previouslyreported to be overexpressed in the protein encoded by the human CUTL1 gene. Although Cux1 is human breast cancers. To investigate its oncogenic potential, expressedin most tissues, Cux2 expression is restrictedprimarily to we engineered two transgenic mouse lines expressing p75 nervous tissues (3). Cux1 knockout mice generally die during the CDP/Cux under the control of the mouse mammarytumor perinatal period, but 20% survive into adulthood. Such surviving virus-long terminal repeat. The FVB strain of mouse is mice displayed phenotypes in various organs including curly generallyused in the generation of mouse models for breast whiskers, growth retardation, delayed differentiation of lung cancer. The transgene was introduced into the hprt locus of epithelia, alteredhair follicle morphogenesis, male infertility, and 129/Ola embryonic stem cells and, following germ line a deficit in T and B cells (6–8). In contrast to the small size of the passage, was backcrossed onto the FVB and C57BL/6 mouse Cux1 knockout mice, transgenic mice expressing Cux1 displayed strains. Here, we describe the phenotype of p75 CDP/Cux multiorgan hyperplasia andorganomegaly (9). Thus, from genetic transgenic virgin female mice of the first backcross gener- studies both in Drosophila andthe mouse, it is clear that the CDP/ ations. We report that after a long latencyperiod, f33% of Cux/Cut gene plays an important role in the development and mice from two independent transgenic lines and from back- homeostasis of several tissues. crosses into either the FVB or the C57BL/6 strains succumbed At least three CDP/Cux protein isoforms can be expressedas the to a similar disease characterized bysplenomegaly,hepato- result of proteolytic processing or transcription initiation at an megaly, and frequent infiltration of leukocytes into non- alternative start site: p200, p110, andp75. The full-length protein, hematopoietic organs like the kidneys and lungs. Although an p200 CDP/Cux, is a complex protein with four evolutionarily excess of B or T cells was observed in three diseased mice, in conserved DNA-binding domains: three Cut repeats (CR1, CR2, and 17 other cases, histologic and flow cytometry analyses CR3) anda Cut homeodomain(Fig. 1; refs. 4, 10–12). The NH 2- revealed the expansion of a population of neutrophils in the terminal endof the full-length protein harbors an autoinhibitory blood, spleen, and bone marrow. The increase in neutrophils domain that inhibits DNA binding (13). Two active transcriptional correlated with signs of anemia and thrombocytopenia, repression domains are present within the carboxyl-terminal whereas there was no indication of a reactive process. domain (R1 and R2; refs. 14–16). The full-length protein was found Therefore, p75 CDP/Cux transgenic mice displayed heightened to be proteolytically processedat the G 1-S transition of the cell susceptibilityto a disease defined as a myeloproliferative cycle, thereby generating the p110 CDP/Cux isoform which disease–like myeloid leukemia. These results indicate that the contains three DNA-binding domains, CR2, CR3, and HD (17). In overexpression of p75 CDP/Cux could alter homeostasis in the addition, a tissue-specific mRNA species was found to code for the hematopoietic compartment. (Cancer Res 2006; 66(19): 9492-501) p75 CDP/Cux isoform which contains only two DNA-binding domains: CR3 and HD (3, 18). Molecular studies showed that the Introduction full-length protein, p200, binds rapidly but transiently to DNA and carries the CCAAT displacement activity (17, 19). In contrast, the The CCAAT-displacement protein/cut homeobox (CDP/Cux) p110 andp75 isoforms behave like classical transcription factors belongs to a family of transcription factors present in all metazoans that engage in slow but stable interactions with DNA (17, 18). CDP/ andis involvedin the control of proliferation anddifferentiation Cux was originally shown to function in precursor cells of various (reviewedin ref. 1). The gene was first identifiedin Drosophila lineages as a transcriptional repressor that down-modulates melanogaster andwas namedafter the ‘‘cut wing’’ phenotype (2). In lineage-specific genes that later become expressedin terminally higher vertebrates, there are two Cut-like genes calledCut-like 1 differentiated cells (1, 20–24). In addition, recent evidence suggests (CUTL1) andCut-like 2 ( CUTL2) in human, andCut-homeobox 1 that the processedisoform can also participate in transcriptional activation andcan stimulate cell proliferation by accelerating entry into S phase (25, 26). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). The I20-mRNA andp75 proteins were foundto be expressed Requests for reprints: Alain Nepveu, Molecular Oncology Group, McGill primarily in the placenta andthymus, but aberrant expression was University, 687 Pine Avenue West, Room H5.21, Montreal, Quebec, Canada H3A 1A1. observedin breast cancer cells (18). In invasive tumors, a Phone: 514-934-1934 ext. 35842; Fax: 514-843-1478; E-mail: [email protected]. I2006 American Association for Cancer Research. significant association was establishedbetween higher I20-mRNA doi:10.1158/0008-5472.CAN-05-4230 expression anda diffuse infiltrative growth pattern (18). In the

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. p75 CDP/Cux Causes a Myeloproliferative Disease present study, we set out to test the hypothesis that p75 CDP/Cux Histology. Tissues were fixed in 4% paraformaldehyde for 24 hours. may play a causal role in cancer. We useda knock-in strategy into Paraffin-embedded sections were then stained with H&E. CD11b+ the hypoxanthine phosphoribosyltransferase (hprt)locusto splenocytes were stainedwith Diff-Quik Stain (DadeBehring, Du ¨dingen, generate transgenic mice containing a p75 CDP/Cux transgene Switzerland). under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) (27). To our surprise, transgenic mice from the first generations of backcrosses to both the FVB and C57BL/6 strains of mouse succumbedto a diseaseinvolving the hematopoietic system. In this report, we characterize the cellular abnormalities encounteredin these transgenic mice.

Materials and Methods Plasmids. Plasmidsequences andmaps will be providedupon request. Expression vectors for CDP/Cux included the complete MMTV-LTR as well as coding sequences for the indicated amino acids of the human CDP/Cux protein: 1-1505, 817-1505, and1062-1505 (GenBank accession no., M74099). For introduction into embryonic stem cells, all of the above constructs were directionally inserted into a slightly modified version of the hprt targeting vector, pMP8SKB (a gift from Sarah Bronson, Pennsylvania State University, Hershey, PA; ref. 27). Cell culture and electroporation. BK4 embryonic stem cells were originally derived from the 129/Ola strain and contain a deletion within the hprt locus that prevents expression of the hprt gene (28). Cell culture and electroporation were done as previously described (29). PCR analysis on genomic DNA was done using a forward primer from the 5¶-flanking hprt genomic sequences (5¶-ggcagaagtagaattaggcttttcagg-3¶) anda reverse primer from the MMTV-LTR sequence (5¶-caaccccttggctgcttctcc-3¶). Generation of transgenic mice. All experiments involving animals were conducted in accordance with the McGill University Animal Care Guide- lines. Targetedembryonic stem cells were injectedinto C57BL/6-derived blastocysts that were then transplantedinto the uteri of recipient females. Resulting chimeric males were bredwith C57BL/6 females, andthe F1 agouti female offspring were backcrossedwith C57BL/6 males. Genotyping was done by PCR analysis of genomic DNA prepared from mouse tail biopsy using a forwardprimer from the MMTV-LTR sequence anda reverse primer from the CDP/Cux sequence. Two lines of p75 CDP/Cux transgenic mice were generated, p75-48 and p75-50, each from an independent blastocyst microinjection with different embryonic stem cell clones. Because the transgene was integratedinto the hprt locus on chromosome X, the transgene wouldbe expectedto be expressedin f50% of the cells in females andin 100% of cells in males. Statistics on penetrance were obtainedwith female mice exclusively. Monitoring mice. Mice were palpatedevery week for the development of ascites. Premoribundmice were anesthetizedwith isofluorane and euthanizedby cervical dislocation.Hematopoietic organs (spleen, lymph nodes, thymus, and bone marrow), liver and lungs were harvested, weighed, andanalyzedby histology, flow cytometry, reverse transcription-PCR (RT-PCR), andWestern blot. The measurement of hematologic variables was done upon sacrifice (Diagnostic Laboratory, Animal Resource Center, McGill University) andbloodsmears were stainedwith a Wright stain.

Figure 1. Generation of p75 CDP/Cux transgenic mice by specific transgenesis. A, expression of CDP/Cux transgenes in tissue culture. Expression vectors were prepared with the MMTV-LTR and the coding sequences for p200, p110, and p75 CDP/Cux. Nuclear extracts were prepared from 293 cells transfected with the MMTV-p75, p110, or p200 plasmids and Western blot was done using the CDP/Cux 1300 antibody. Bottom, diagrams showing the three CDP/Cux isoforms, p200, p110, and p75 and their evolutionarily conserved domains: CC, coiled-coil; CR1, CR2, and CR3, Cut repeats 1, 2, and 3; HD, homeodomain, R1 and R2, repression domains 1 and 2. B, integrated transgene. The p75 CDP/Cux isoform, under the control of the MMTV-LTR promoter, was specifically integrated into the hprt locus on the X chromosome. C, expression of the p75 CDP/Cux transgene in healthy transgenic mice. RNA was prepared from various tissues of a 12-month-old healthy transgenic mouse from 129/Ola:C57BL/6 backcross 1, and submitted to RT-PCR analysis using primers specifically amplifying the transgene. Bottom, diagram showing the position of the sense and reverse primers. www.aacrjournals.org 9493 Cancer Res 2006; 66: (19). October 1, 2006

RT-PCR. RNA was preparedusing TRIzol (Invitrogen, Carlsbad,CA) and Generation of p75 CDP/Cux transgenic mice byspecific cDNA was preparedusing the Superscript II RNase H-reverse transcriptase transgenesis. To avoidcomplications resulting from variations in kit (Invitrogen). Real-time PCR was done on a LightCycler (Roche, Basel, copy number andintegration site effects, we usedthe methodof Switzerland) using the FastStart DNA Master SYBR Green kit (Roche). targetedtransgenesis to insert the construct into the mouse hprt Preparation of protein extracts. Total protein extracts were prepared locus (27). To confirm integration by homologous recombination, by homogenizing tissue in NP40 buffer [150 mmol/L of NaCl, 50 mmol/L of ¶ Tris (pH 8.0), 1% NP40, 10% glycerol, 0.5 mmol/L of DTT, protease inhibitor PCR analysis was done using a forward primer from the 5 -flanking mix tablet (from Roche)] andmixing for 30 minutes at 4 jC. The extracts hprt genomic sequences anda reverse primer from the MMTV-LTR were then centrifugedfor 15 minutes at 4 jC andthe supernatants collected. sequences (Fig. 1B). Two independent lines of p75 CDP/Cux Nuclear extracts were preparedas describedpreviously (17). transgenic mice were generated, p75-48 and p75-50. In previous Flow cytometry analysis. Single cell suspensions were preparedfrom studies, development and cancer of the mammary gland has bone marrow, spleen, lymph nodes, thymus, blood, and infiltrated organs generally been analyzedin the FVB strain of mouse (36, 37). (liver andlung), andRBC were lysedwith ACK buffer (0.15 mol/L of Therefore, we initiateda series of backcrosses to the FVB strain NH4Cl, 1 mmol/L of KHCO3, 0.1 mmol/L of Na2-EDTA; adjusted to pH and, as a control, the C57BL/6 strain. As the backcrosses were j 7.2-7.4). Livers were incubatedfor 20 minutes at 37 C with collagenase under way, a high proportion of mice from the first generations, prior to preparation of single cell suspensions. Cells were then resuspended both from the FVB andC57BL/6 backcrosses, succumbedto what in 37% Percoll andcentrifugedto isolate hematopoietic cells. From the single cell suspensions obtained, 106 cells were incubatedfor 15 minutes on seemedto be a similar diseasecharacterizedby splenomegaly ice in blocking solution (2.4G2) andwere then stainedwith monoclonal andinfiltration of WBC to other organs (Table 1; Supplemental antibodies conjugated with phycoerythrin, FITC, or biotin, to detect Table S1). In contrast, the p110 transgenic mice did not display a either myeloidcells, B lymphocytes, or T lymphocytes. The following higher incidence of this disease (Table 1). Here, we describe the antibodies, obtained from BD PharMingen (Palo Alto, CA) and Cedarlane phenotype of the p75-transgenic mice. (Hornby, ON, Canada), were used: CD11b (Mac-1), Gr-1, 7/4, CD4, CD8, B220, Expression of the p75 CDP/Cux transgene in several tissues. IgM, F4/80, andCD11c. Cells were submittedto a FACScan flow cytometer A 10-month-oldhealthy virgin female transgenic mouse from the (Becton Dickinson, Franklin Lakes, NJ) anddatawere analyzedwith backcross 1 (BC1) generation to C57BL/6 was sacrificedand FLOWJO software developed by Tree Star (San Carlos, CA). transgene expression was investigatedby RT-PCR (Fig. 1 C). We Immunoblotting. Western blot analyses with actin (Santa Cruz observedelevatedexpression of the transgene in the spleen, kidney, Biotechnology, Santa Cruz, CA; 1/2,000) and1300 (1/1,000) were doneas previously described (17). andbrain, andrelatively lower expression in the liver (Fig. 1 C, lanes Isolation of CD11b-positive splenocytes. Splenocytes were stained 2, 4, 5, and 7). Because the mouse hadnot been subjectedto with CD11b-biotin antibody after RBC lysis with ACK. The stained cells perfusion, we cannot exclude that the expression detected in some were applied on streptavidin magnetic beads (MACS) for 15 minutes and tissues, in particular in the liver andbrain, was not dueto blood then passedthrough a magnetic column (MACS). Greater than 97% of the contamination. Considering that the transgene was integrated into cells recovered were CD11b-positive. TRIzol was added to these cells to the hprt locus andthat one chromosome X is inactivatedin each extract mRNA andfor RT-PCR analyses. cell, in females, f50% of the cells wouldbe expectedto express the Transplantation. Ten million splenocytes were transplantedinto transgene in those tissues showing expression. sublethally irradiated (650 rad) immunocompromised nude mice. Recipient A fraction of p75 CDP/Cux transgenic mice developed mice were followedby observation of their health status andalso by blood hepatosplenomegaly. A cohort of p75 CDP/Cux transgenic (n = 60) test analyses. andwild-type mice ( n = 35) were kept until moribund. Mice were palpatedevery week for the developmentof ascites andenlarged Results abdomen. A fraction of transgenic mice (20 of 60) developed what The p75 CDP/Cux isoform can be expressed from the seemed to be a hematopoietic disorder characterized by hepatos- MMTV-LTR. The p110 andp75 CDP/Cux isoforms were previously plenomegaly (Fig. 2; Supplemental Table S1). The enlargedspleens reportedto be aberrantly expressedin uterine leiomyomas and of diseased mice weighed, on average, 18.8 times that of normal breast tumors, respectively (18, 30). To assess the oncogenic spleens (Table 2). The latency was fairly short in two cases potential of p110 andp75 CDP/Cux, we set out to generate (9 months) but was generally much longer with an average of transgenic mice that wouldexpress these isoforms specifically in 20 months. Overall, mice from backcrosses 1 to 3 in C57BL/6, and mammary epithelial cells. We generatedan expression vector from backcross 1 in FVB, developed the disease with a penetrance carrying the coding sequences for p110 or p75 CDP/Cux of 33% (20 of 60) andan average latency of 20.3 months (Table 1). downstream of the MMTV-LTR. These regulatory sequences were Note that statistics on penetrance were obtainedwith female mice previously shown to drive expression in mammary epithelial cells, exclusively. In comparison, nontransgenic littermates developed the but transgene expression has been reportedin other cell types, disease with a penetrance of 8.6% (3 of 35 mice) and an average latency particularly in the hematopoietic system (31–33). To investigate of 20.5 months (Table 1). Therefore, the susceptibility to this disease expression of the three CDP/Cux isoforms under the control of the was increasedapproximately four times in p75 transgenic mice. MMTV-LTR, the corresponding expression plasmids were intro- Splenomegaly was caused by myeloid hyperplasia. Histologic duced by transfection into 293 cells, nuclear protein extracts were analysis revealedthat the enlargedspleens of transgenic mice did preparedandWestern blot analysis was doneusing the CDP/Cux not maintain a normal architecture. Regions of white pulp were antibody 1300 (Fig. 1A). Both the p75 andp110 isoforms were compressed, whereas there was a large number of cells resembling expressedin 293 cells, but little, if any, p200 expression was detected polymorphonuclear leukocytes (Fig. 2B). Indeed, ring-shaped nuclei (Fig. 1A, lanes 1-3). We consider it likely that the role of p200 as a characteristic of neutrophils were observedfollowing the purifica- transcriptional repressor of MMTV-LTR precludes its expression tion of CD11b-positive cells andstaining with Diff-Quik (Fig. 2 D). from the same regulatory sequences (34, 35). However, our results In three cases (mice p75-48-54, p75-50-116, andp75-50-129), an clearly showedthat both the p110 andp75 CDP/Cux isoforms can increasednumber of both granulocytes andmegakaryocytes was be expressedusing the MMTV-LTR regulatory sequences. observedin the spleen (Fig. 2 D).

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Table 1. Disease penetrance and latency in p75 CDP/Cux transgenic mice

Strain Transgene status Total number Mice with hematopoietic Average Penetrance (%) of mice disorders latency (mo)

All mice p75 CDP/Cux 60 20 20.3 33.3** Wild-type 35 3 20.5 8.6 p75-48 line p75 CDP/Cux 38 13 20.8 34.2* p75-50 line p75 CDP/Cux 22 7 18.2 31.8* BC1 129/Ola p75 CDP/Cux 15 8 17.8 53.3* Â C57BL/6 Wild-type 14 2 19.5 14.0 BC2 129/Ola p75 CDP/Cux 26 7 21.1 26.9ND Â C57BL/6 Wild-type — — — BC3 129/Ola p75 CDP/Cux 9 2 19.5 22.0NS Â C57BL/6 Wild-type 13 1 21.0 7.7 BC1 129/Ola p75 CDP/Cux 10 3 26.0 30* Â FVB Wild-type 8 0 — 0 All p110 mice p110 CDP/Cux 63 4 22.5 6.3NS Wild-type 23 1 22.0 4.3

NOTE: Latency (age of death) and penetrance (number of sick transgenic mice/total number of transgenic mice) were calculated for all transgenic mice, for each of the two transgenic lines (p75-48 andp75-50), andfor each of the backcrosses. Note that all mice were females. Considering that the transge ne was integratedinto the hprt locus andthat one chromosome X is inactivatedin each cell, the transgene wouldbe expectedto be expressedin 50% of the cells. Using the two-proportion Z test, the difference in penetrance between p75 transgenic and wild-type littermates was found to be significant (*, P V 0.05) or very significant (**, P V 0.01). In contrast, the difference in penetrance between p110 transgenic and wild-type littermates was not significant. Abbreviations: NS, not significant; ND, not determined.

Nonhematopoietic organs were infiltrated in manysick The expanded cell population expressed neutrophil surface mice. When observedat autopsy, the lungs andlivers of sick mice markers. A large proportion of cells extractedfrom the spleen, were of unusual color andtexture, suggesting infiltration by bone marrow, liver, andbloodof transgenic mice displayed another cell type (Fig. 2A; Supplemental Table S1). Histologic increasedsize andgranulosity as shown from sideandforward examination confirmedthe presence of an overrepresented scatter analyses (Supplemental Fig. S1). Flow cytometry analysis population of cells in the liver, lungs, kidneys, and blood (Fig. 2B was done on 15 affected transgenic mice and 16 wild-type and C). In contrast, in most cases, the thymus andlymph nodesof littermates using antibodies specific for cell surface markers of sick mice seemednormal upon morphologic andhistologic myeloidcells (CD11b, 7/4, andGr-1), B lymphocytes (B220, IgM), examinations, andexhibitednormal staining patterns in flow andT lymphocytes (CD4, CD8; Table 2; Supplemental Fig. S1; cytometry analysis (data not shown). Supplemental Table S2). With the exception of two mice that Anemia and thrombocytopenia in sick transgenic mice. presented with a hematopoietic disorder involving the lymphoid Hematologic variables were measuredin the peripheral bloodof compartment (p75-48-129 andp75-48-136, see below), most sick affectedtransgenic andwild-type littermate mice (Table 3; Fig. 2 C). transgenic mice displayed an excess of CD11b-positive cells in the Although the hemoglobin concentration, hematocrit, andnumber spleen, bone marrow, liver, andblood.In contrast, B220/IgM- of RBC andplatelets were significantly decreasedin sick transgenic positive cells were underrepresented in the spleen, bone marrow, mice, the number of WBC was significantly increased. In most sick andliver of diseasedtransgenic mice. CD11b/Gr-1/7/4 triple transgenic mice, the increase in WBC resultedfrom an increase in staining revealeda large overrepresentation of neutrophils in the the number of neutrophils. Morphologic analysis of bloodsmears spleen andbloodof diseasedtransgenicmice (Table 2; Supple- did not reveal the presence of progranulocytes, myelocytes, mental Fig. S1D). Moreover, in the spleen, we observedboth low metamyelocytes, or bandcells. Myeloblasts were detectedinonly andhigh Gr-1 expression levels among these cells. These findings one sick transgenic mouse (Table 3, mouse #116). Lymphopoiesis suggest that the overrepresentedcells are neutrophils andthat did not seem to be impaired in affected transgenic mice because complete maturation can occur in this cell lineage. Altogether, data the number of lymphocytes did not vary significantly. However, from histologic andflow cytometry analyses indicatedthatmost of because the number of neutrophils was increased, the percentage the affectedp75 CDP/Cux transgenic mice displayedanoverrep- of neutrophils in the bloodwas significantly increasedat the resentation of neutrophils in the blood, spleen, liver, and bone expense of the percentage of lymphocytes. In summary, the analysis marrow, andin many cases, in nonhematopoietic organs like the of bloodsmears revealedthat affectedtransgenic mice suffered kidneys and lungs. from anemia andthrombocytopenia, whereas their bloodshowed The disease is not transplantable. Splenocytes from seven sick an increase in the number of neutrophils. transgenic mice were transplantedinto sublethally irradiated www.aacrjournals.org 9495 Cancer Res 2006; 66: (19). October 1, 2006

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Cancer Research immunocompromisednudemice. After a periodof 10 to 13 one mouse showedboth an excess of myeloidcells in the spleen months, no recipient has yet developed a hematopoietic disorder. anda grossly enlargedlymph nodecontaining mostly B cells À We conclude that the disease is not transplantable. (B220+IgM ; Table 2, p75-48-73; data not shown). Other less frequent hematopoietic disorders in p75 CDP/ The p75 CDP/Cux transgene is expressed in the spleen, liver, Cux transgenic mice. A few transgenic mice succumbedto and CD11b+ cells of leukemic transgenic mice. The transgene different hematopoietic disorders. The presence of peripheral mRNA was expressedin the spleen andliver of transgenic mouse myeloblasts in the bloodsmear of a transgenic mouse suggesteda andin CD11b + cells purifiedfrom the spleen (Fig. 3 A, lanes 2, 4, blast transformation (Table 3, p75-50-116). Two transgenic mice and 5). Transgene protein expressions were investigatedin four seemedto suffer from a disorder involving the lymphoid groups of healthy or leukemic transgenic mice andtheir respective compartment: flow cytometry analysis of the spleen revealedan wild-type littermates. The p75 protein was observed in three overrepresentation of CD4-positive cells in one mouse (Table 2, leukemic transgenic mice, but very little or no expression was p75-48-129), whereas examination of spleen sections revealedthe detected in the wild-type littermates or in the healthy transgenic presence of a large number of atypical lymphocytes with numerous mice (Fig. 3B, leukemic transgenic mice in lanes 2, 5, and 7; wild- mitotic figures in the other (p75-48-136, data not shown). Finally, type littermates in lanes 1, 3, 6, and 8; healthy transgenic mouse in

Figure 2. p75 CDP/Cux mice develop splenomegaly and display infiltration of various organs by hematopoietic cells. A, hepatosplenomegaly in a 12-month-old p75 CDP/Cux mouse with a hematopoietic disorder as well as enlarged spleen, lung, and liver due to hematopoietic infiltrate. Pictures show the typical phenotype observed in transgenic mice that developed this disorder. B to D, histologic examination of various organs in diseased mice. Tissue sections from a diseased transgenic mouse and a wild-type littermate were stained with H&E (original magnifications, Â100 and Â400): spleen, liver, lungs, and kidneys (B). C, blood vessel in the transgenic mouse liver showing an overrepresentation of leukocyte (H&E staining) and peripheral blood smears comparing a transgenic mouse (p75-50-164) to its wild-type littermate (Wright staining). D, Diff-Quik–stained cytospin preparation of CD11b+ myeloid cells sorted on magnetic beads from an enlarged spleen, showing an overrepresentation of neutrophils and H&E staining of spleens from transgenic and wild-type littermate (arrows, megakaryocytes).

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Table 2. Flow cytometry analysis of spleens

Mouse ID B220+ IgM+ CD4+ CD8+ CD11b+ Gr1+low/7/4+ Gr-1+high/7/4+ CD34+ Spleen Liver (%) (%) (%) (%) (%) (%) (%) (%) weight (g) weight (g)

Transgenic mice with normal organs p75-48-36 59.6 53.7 22.2 11.2 36.5 14.1 9.3 — 0.18 — p75-50-84 44.9 43.4 — — 29.1 — — — 0.07 1.61 p75-48-103 — — 12.3 6.2 52.3 — 30.6 — 0.20 — p75-48-106 44.2 40.7 14.8 4.6 40.7 49.5 21.7 — 0.18 — p75-48-137 39.2 38.2 12.2 12.8 37.4 9.0 10.5 5.2 0.30 1.95 p75-50-149 43.0 40.7 17.9 10.9 22.4 — — — 0.18 1.90 p75-50-209 44.8 41.3 21.1 17.8 12.4 11.3 20.7 — 0.10 — p75-50-221 — — — — 12.2 8.6 15.5 — 0.11 — Average ratio 0.9NS 0.9NS 1.0NS 1.2NS 2.2* 1.2NS 0.8NS 1.0ND 1.7* 1.1NS transgenic/control Transgenic mice with enlargedorgans p75-48-27 27.2 — 5.8 5.6 45.5 — — — 4.93 — p75-48-37 25.1 12.6 12.8 3.7 51.8 — 61.8 — 4.80 3.22 p75-48-48 16.9 9.5 9.6 2.8 36.0 — 49.7 32.1 0.76 4.63 p75-48-54 7.3 4.3 4.8 8.2 88.9 4.5 21.1 — 1.39 5.15 p75-48-55 12.3 7.7 16.0 8.0 35.4 — 73.2 — 3.73 — p75-50-64 9.3 7.2 16.3 4.3 84.7 32.7 56.5 4.1 0.86 — p75-48-73 28.0 20.4 12.8 2.3 33.1 35.8 49.4 — 0.33 — p75-50-116 16.3 15.2 11.9 2.3 60.9 38.7 36.5 — 1.01 — p75-48-129 14.6 1.6 39.8 3.0 11.4 — 10.3 4.1 0.96 1.89 p75-50-129 14.0 12.3 4.0 1.7 61.5 38.5 46.6 — 0.70 — p75-48-136 1.0 32.4 19.4 10.6 13.0 15.1 28.3 1.8 2.12 4.42 p75-48-138 14.2 8.3 8.2 3.5 48.1 41.1 50.9 — 1.38 — p75-48-139 27.3 21.5 16.0 4.4 43.7 40.2 36.3 — 0.84 — p75-50-141 32.8 29.0 8.8 3.9 53.6 32.4 26.1 — 0.40 — p75-50-164 2.0 2.0 7.2 2.1 47.4 38.1 29.0 13.9 3.95 2.62 Average ratio 0.3*** 0.3*** 0.8NS 0.5*** 3.4*** 6.9** 1.9** 2.2NS 18.8*** 2.28* transgenic/control Control mice (n = 16) 52 F 10 46 F 617F 59F 314F 616F 10 22 F 12 5 F 1(n =4) 0.1F 0.1 1.6 F 0.5

NOTE: Cells were isolatedfrom the spleen of transgenic mice with normal-sizedspleen andfrom transgenic mice with an enlargedspleen, andstained for flow cytometry analysis. Numbers of Gr1+low/7/4+ andGr1 +high/7/4+ cells are calculatedas a percentage of CD11b + cells. Ratios of transgenic values over control values were calculatedandaveraged.Statistical significance was verifiedusing a t test (*, P V 0.05; **, P V 0.01, or ***, P V 0.001). Abbreviations: NS, not significant. ND, not determined.

lanes 4 and 9). These results suggest that the development of the confirmedthat p27 mRNA levels were reducedin the spleen and disease is associated with increased expression of the p75 CDP/ CD11b+ cells of all leukemic mice. Cux protein. Expression of CDP/Cux target genes in the spleen and CD11b+ cells of leukemic mice. Previous studies have identified a Discussion number of CDP/Cux putative targets, some with relevance to Our study revealed that p75 CDP/Cux transgenic mice display cancer (9, 25, 38, 39). Total RNA was preparedfrom the enlarged increasedsusceptibility to a myeloproliferative disease.In investi- spleen of four leukemic mice andfrom CD11b + cells purifiedfrom gating the etiology of the disease, it was particularly important to the enlargedspleens of two leukemic mice. As a control, total RNA determine whether the disease was the result of reactive conditions was preparedfrom the spleen of a wild-typelittermate. However, as opposedto a neoplastic process. Several criteria enabledus to because the spleen of leukemic mice containeda higher proportion exclude that an inflammatory response was responsible for the of CD11b cells than that from normal mice, as a secondcontrol, we expansion of the CD11b/Gr-1 compartment in various organs. First, preparedRNA from CD11b + cells isolatedfrom a pool of spleens cells of affectedorgans were predominantlyof granulocytic lineage, from five wild-type C57BL/6 mice. Changes in the expression levels whereas reactive myeloidhyperplasia is characterizedby a of some genes were detected, however, apart for the p27 CDK heterogeneous population of myeloid, erythroid, and megakaryo- inhibitor, no change in expression was consistently observedin all cytic cells. Second, in contrast to reactive processes, in many leukemic mice. Interestingly, the levels of p27 mRNA seemedto be transgenic mice, CD11b/Gr-1 cells spreadto the liver andcaused decreased in the spleen and CD11b+ cells of all leukemic mice its enlargement, andsometimes spreadto some nonhematopoietic (Fig. 3C). Semiquantitative real-time PCR analysis was done to organs like the lungs and kidneys. Third, the disease developed verify mRNA levels for both p27 andp21 (Fig. 3 D). The results without apparent contribution from any environmental factor. www.aacrjournals.org 9497 Cancer Res 2006; 66: (19). October 1, 2006

Nonhematopoietic tumors were absent from the affectedmice. The Fig. S1; Supplemental Table S2). The absence of a block to mice were maintainedin a specific pathogen–free environment differentiation was also in agreement with the slow progression of andwere not exposedto drugs or toxins. Transgenic and the disease and, except for one case, the absence of an acute phase nontransgenic littermates were exposedto identicalenvironmental (data not shown). In addition, we note that the disease had not conditions because they were maintained in the same cage yet, developed 10 months after the transplantation of neoplastic only 3 of 35 nontransgenic mice developed disease. Moreover, the myeloidcells into sublethally irradiatedhistocompatible recipients. latency was significantly longer in mice obtainedfrom the FVB Altogether, these criteria would define the disease as a myelopro- versus the C57BL/6 backcross populations, further supporting the liferative disease–like myeloid leukemia. genetic basis of the disease. Although the diseased p75 mice did not exhibit the typical The Bethesda proposals for the classification of nonlymphoid cytogenetic rearrangements involving the BCR/ABL oncogene, the hematopoietic neoplasms in mice provides precise criteria for myeloproliferative disease in p75 CDP/Cux transgenic mice shares accurate diagnosis (40). The disease that developed most often in several features with chronic myelogenous leukemia (CML) in p75 CDP/Cux transgenic mice meets several of the criteria that humans. In particular, not only did the disease evolve slowly, but define a nonlymphoid leukemia. The disease diffusely involved except for two mice that became moribundat 9 months of age, it hematopoietic tissues with an increase in myeloidcells in both the affected old individuals without appreciably shortening their life spleen andbone marrow, andwas accompaniedby anemia and spans. In this respect, the p75 CDP/Cux transgenics might prove to thrombocytopenia (Fig. 2; Tables 2 and3; Supplemental Tables S1 be a useful mouse model to study some aspects of CML. In addition andS2; Supplemental Fig. S1). Myeloidcells were often dissemi- to a BCR-ABL transgenic line in which p210 was driven by the tec natedin nonhematopoietic organs includingthe liver, lungs, and promoter, three knockouts were also foundto exhibit a CML-like kidneys (Fig. 2). Leukocytosis was present except for one exception phenotype. Inactivation of either the IFN consensus sequence (p75-48-48, 32% CD34+ cells in spleen), nonlymphoidimmature binding protein (ICSBP), JunB or the estrogen receptor h (ERh) forms/blasts did not make up 20% of leukocytes in the peripheral invariably resultedin a CML-like diseasethat is characterizedby an blood, spleen, or bone marrow (Tables 2 and 3; Supplemental Table elevation of neutrophils in hematopoietic tissues (refs. 41–44, S2; data not shown). Indeed, myeloid cells did not lose their reviewedin ref. 45). The latency was short for ICSBP andJunB capacity to differentiate as shown by the presence of CD11b/7/4 knockouts, but latency was long for the ERh knockout. One cells with high Gr-1 expression levels (Table 2; Supplemental important difference between these mouse models and the

Table 3. Peripheral blood analyses and blood differentials in p75 CDP/Cux transgenic mice

Mouse ID Group no. Fold difference Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

WT 64 WT 54 55 WT 48 WT 37 WT 141 129 116 WT 164

Hematocrit (L/L) 0.44 0.27 0.35 0.07 0.16 0.40 0.32 0.28 0.40 0.49 0.36 0.22 0.17 0.39 0.38 0.7 F 0.2* Hemoglobin (g/L) 153 89 133 27 53 135 99 103 130 169 113 72 56 141 118 0.6 F 0.2** RBCs Â 1012/L 9.8 5.1 7.8 0.8 2.3 9.1 4.9 6.7 8.6 11.2 8.0 3.8 3.6 9.0 6.3 0.6 F 0.1** Platelets Â 109/L 1043 632 681 100 65 426 55 507 361 1053 1250 953 1230 690 157 0.6 F 0.5* WBC Â 109/L 5.8 10.6 2.3 4.6 5.7 3.6 2.6 4.0 5.4 6.0 5.3 10.4 14.0 2.4 16.0 2.1 F 0.9* Neutrophils (%) 31 62 42 62 68 44 65 24 18 16 46 64 54 15 68 2.5 F 1.4** Bands (%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NS Metamyelocytes (%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NS Myelocytes (%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NS Myeloblasts (%) 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 NS Lymphocytes (%) 69 37 57 37 31 46 35 75 78 82 53 35 39 82 20 0.7 F 0.2** Monocytes (%) 0 1 1 1 1 8 0 0 4 2 1 1 0 3 10 0.9 F 0.2NS Eosinophils (%) 0 0 0 0 0 2 0 0 0 0 0 0 0 — 2 NS Basophils (%) 0 0 0 0 0 0 0 0 0 0 0 0 0 — 0 NS Neutrophils Â 109/L 1.8 6.6 1.0 2.9 3.9 1.6 1.7 1.0 1.0 1.0 2.4 6.7 7.3 0.4 6.6 4.4 F 2.6* Myeloblasts Â 109/L 0 0 — 0 — — 0 0 0 — 0 0 0.7 — 0 NS Lymphocytes Â 109/L 4.1 3.9 1.3 1.7 1.8 1.7 0.9 3.0 4.2 4.9 2.8 3.6 5.3 2.0 3.2 1.2 F 0.5NS Monocytes Â 109/L 0 0.10 0.02 0.05 0.06 0.30 0 0 0 0.12 0.05 0.10 0.30 0.07 1.60 NS Eosinophils Â 109/L 0 0 0 0 0 0.07 0 0 0 0 0 0 0 0 0.32 NS

NOTE: Differential morphologic analysis of peripheral bloodcells was doneon May-Gru¨nwald-Giemsa–stained blood smears from sick transgenic mice and matched normal littermates (WT). Cells were counted and assigned according to morphologic criteria detailed in ref. 49. Average fold difference: the fold difference was first measured between each transgenic mouse and its wild-type littermate, the average fold difference was then calculated and statistical analysis of the values was done using a t test. *, P V 0.05, difference is significant; **, P V 0.01, difference is very significant. Abbreviation: NS, not significant.

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Figure 3. Expression of the p75 CDP/Cux transgene and putative targets in the spleen, liver, and CD11b+ cells of leukemic mice. A, RNA was prepared from the spleen, liver, and CD11b+ sorted cells of the leukemic mouse p75-48-37 and a wild-type littermate. RT-PCR analysis was done using primers specific for the p75 transgene or glyceraldehyde-3-phosphate dehydrogenase as a control. B, four groups of littermates (A, B, C, and D) including wild-type mice (wt), healthy, and diseased transgenic mice (tg) were sacrificed at 18, 12, 17.5, and 8 months, respectively. Total protein extracts were obtained from the spleen and 100 Agof protein was subjected to Western blot analysis using CDP/Cux antibody 1300 (see Fig. 1A), or anti-actin antibody. C, expression of h-actin and of putative transcriptional targets of CDP/Cux was studied by RT-PCR analysis using RNA from the following sources. RNA was purified either from the whole spleen (sp) or from CD11b-positive cells isolated from the enlarged spleens of individual transgenic mice or from a pool of spleens from five wild-type C57BL/6 mice. D, quantitative real-time PCR analysis was done for p27 and p21 using the indicated samples as in (A).

transgenics described here is that the inactivation of these genes 60) transgenic mice were virgin, contributedto the weak occurredearly, affectedall cells, andhadan effect on the behavior expression in mammary glands. As MMTV-directed transgene of hematopoietic stem cells, whereas the p75 CDP/Cux transgene experiments have been done in the FVB strain, we expect that in was expressedin only half of the cells andlater in ontogeny, most future backcrosses, transgene expression in mammary glands will likely within a committedmyeloidprogenitor. Future studies augment in parallel with the FVB genetic component andwill shoulddetermine whether there are common transcriptional further increase in multiparous females (32, 33). This said, MMTV- targets or signal transduction pathways operating in p75 CDP/ dependent transgene expression alone cannot explain the observed Cux or BCR-ABL-expressing cells, andin ICSBP, JunB, or ER h phenotypes. We consider that the heightened susceptibility to a knockout mice. In light of the phenotypic similarities in such mice, myeloproliferative disease in p75-transgenic mice likely reveals a it is possible that CDP/Cux, ICSBP, JunB, andER h are involvedin a particular tropism of the p75 CDP/Cux isoform towards certain common transcriptional regulatory network or even function as myeloidprecursor cells. This notion is reinforcedby the direct regulators of one another. Also, it remains to be determined contrasting small incidence of myeloproliferative diseases that if up-regulation of p75 CDP/Cux transcription or activity is an developed in MMTV-p110 CDP/Cux mice (Table 1). We note that etiologic factor in human CML, in particular, among the 5% of the p75 CDP/Cux transgene was expressedweakly, if at all, in the patients who carry a diagnosis of CML but do not harbor the spleen of healthy transgenic mice andthat diseasewas associated Philadelphia (Ph) chromosome. with increasedexpression of the transgene. This indicatesthat Our finding that MMTV-p75 CDP/Cux transgenic mice devel- increasedp75 expression was selectedfor, andprobably playeda oped a myeloproliferative disease was unexpected. We believe two causative role, in the neoplastic process. The increase in p75 reasons explain the low incidence of tumors in mammary glands: expression observedin affectedtransgenic mice also suggests that the weak expression of the transgene in mammary glands and the another event was requiredfor the activation of transgene apparent cell type–specific effect of p75 CDP/Cux. The genetic expression. It will be important to determine the cell type in backgroundof transgenic mice, andthe fact that almost all (56 of which this event takes place. The failure to transplant the disease www.aacrjournals.org 9499 Cancer Res 2006; 66: (19). October 1, 2006

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Cancer Research suggests that the event leading to the activation of p75 transgene whether this spectrum of diseases reflects the tropism of the p75 expression did not occur in a hematopoietic stem cell but, most protein or the cell type specificity of transgene expression. Future likely, in a committedmyeloidprogenitor. This couldbe the reason studies should investigate the phenotype resulting from the for the long latency periodandwouldfurther suggest that the expression of CDP/Cux isoforms in early hematopoietic precursor ability to transplant anda shorter latency couldbe obtainedby or stem cells. combining p75 CDP/Cux with a promoter that enables expression in the hematopoietic stem cell or an early myeloidprogenitor. Indeed, several studies have shown that the phenotype of murine Acknowledgments transgenic models of human leukemia is critically dependent on Received12/1/2005; revised5/12/2006; accepted7/13/2006. the cellular compartment that is targeted(reviewedin refs. 46–48). Grant support: Canadian Breast Cancer Research Alliance grant no. 014348 (A. Nepveu) andthe Regulatory Genetics Program of Genome Quebec/Genome For example, the type of myeloproliferative diseases that were Canada (A.C. Peterson). generated depended on whether the inactivation of JunB or 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 transduction of BCR-ABL occurred in hematopoietic stem cells or with 18 U.S.C. Section 1734 solely to indicate this fact. in granulocyte-macrophage progenitor cells (41, 48). Most of the A. Nepveu andS. Fournier are the recipients of scholarships from the Fondsdela hematopoietic disorders that developed in the cohort of MMTV- Recherche en Sante´du Que´bec.C. Cadieux is the recipient of studentships from the Royal Victoria Hospital Research Institute (2005) andfrom the Department of Defense p75 transgenic mice involvedmyeloidcells; however, in a few mice, Breast Cancer Research Program (2006). the lymphoidcompartment was affected.It is not clear at this point We acknowledge the expertise of Ms. Jo-Ann Bader for the histological work.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Transgenic Mice Expressing the p75 CCAAT-Displacement Protein/Cut Homeobox Isoform Develop a Myeloproliferative Disease −Like Myeloid Leukemia

Chantal Cadieux, Sylvie Fournier, Alan C. Peterson, et al.

Cancer Res 2006;66:9492-9501.

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