Proc. Natl. Acad. Sci. USAVol. 93, pp. 7900-7904, July 1996Medical Sciences

Transgenic expression of PML/RARa impairs myelopoiesis(acute promyelocytic leukemia/transgene/all-trans retinoic acid)

ELLEN EARLY*, MALCOLM A. S. MOOREtt, AKIRA KAKIzuKA§1, KATHRYN NASON-BURdHENAL*,PATRICK MARTIN* $t, RONALD M. EvANS§, AND ETHAN DMITROVSKY* ** tt*Department of Medicine, Laboratory of Molecular Medicine, **Molecular Pharmacology and Therapeutics Program, and tLaboratory of DevelopmentalHematopoiesis, *Cell Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and §Gene ExpressionLaboratory, The Salk Institute for Biological Studies and Howard Hughes Medical Institute, San Diego, CA 92186

Contributed by Ronald M. Evans, April 17, 1997

ABSTRACT The translocation found in acute promyelo-cytic leukemia rearranges the promyelocytic leukemia gene(PML) on chromosome 15 with the retinoic acid receptor a(RARa) on chromosome 17. This yields a fusion transcript,PML/RARa, a transcription factor with reported dominantnegative functions in the absence of hormone. Clinical remis-sions induced with all-trans retinoic acid (RA) treatment inacute promyelocytic leukemia are linked to PML/RARaexpression in leukemic cells. To evaluate the PML/RARa rolein myelopoiesis, transgenic mice expressing PML/RARa wereengineered. A full-length PML/RARa cDNA driven by theCD11b promoter was expressed in transgenic mice. Expres-sion was confirmed in the bone marrow with a reversetranscription PCR assay. Basal total white blood cell andgranulocyte counts did not appreciably differ between PML/RARa transgenic and control mice. Cell sorter analysis ofCD11b+ bone marrow cells revealed similar CD11b+ popu-lations in transgenic and control mice. However, in vitro clonalgrowth assays performed on peripheral blood from transgenicversus control mice revealed a marked reduction of myeloidprogenitors, especially in those responding to granulocyte/macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand co-treatment did not overcome this inhibition. Impaired myelo-poiesis in vivo was shown by stressing these mice withsublethal irradiation. Following irradiation, PML/RAReitransgenic mice, as compared with controls, more rapidlydepressed peripheral white blod cell and granulocyte counts.As expected, nearly all control mice (94.4%) survived irradi-ation, yet this irradiation was lethal to 45.8% ofPML/RARatransgenic mice. Lethality was associated with more severeleukopenia in transgenic versus control mice. Retinoic acidtreatment of irradiated PML/RARci mice enhanced granulo-cyte recovery. These data suggest that abnormal myelopoiesisdue to PML/RARa expression is an early event in oncogenictransformation.

In acute promyelocytic leukemia (APL), the balanced t(15;17)translocation rearranges the promyelocytic leukemia gene(PML) on chromosome 15 with the retinoic acid receptor a(RARa) located on chromosome 17 (1-3). This translocationleads to expression of a fusion transcript, PML/RARa, inAPLcells (4-6). Variant translocations exist and also involverearrangements with RARa but are much less frequent thanthe t(15;17) in APL (7). PML/RARa is co-expressed in APLcells with PML and RARa (4-6). All-trans retinoic acid (RA)treatment of APL patients causes transient complete clinicalremissions in patients whose leukemic cells express PML/RARa (6, 8-10). Successful RA treatment is associated within vivo leukemic cell maturation (9, 10). It is postulated that

PML/RARa functions as a transcription factor with dominantnegative functions exerted on the RARa or PML pathways (4,5, 11, 12). That PML/RARa has an inhibitory role in APLcells is consistent with the finding that APL cells have anaberrant nuclear localization of PML. This abnormality isreversed by RA treatment (13, 14). Direct inhibitory effects ofPML/RARa in myeloid cells was shown through PML/RARaexpression in U937 or HL-60 myeloid leukemic cells (11, 15).In NB4 cells, the sole APL line expressing PML/RARa andinducing a mature myeloid phenotype following RA treatment(16), over-expression of the nonrearranged alleles, RARa, orPML, leads to growth suppression of transfectants (17, 18).This growth suppression is hypothesized to result from antag-onism of PML/RARa function in these APL cells.

Biologic effects of an expressed dominant negative RARain murine myeloid leukemic and progenitor cells have beenreported (19, 20). When endogenous RARa activity is sup-pressed in myeloid leukemic cells by expression of a dominantnegative RARa, granulocyte/macrophage colony-stimulatingfactor (GM-CSF)-induced neutrophil differentiation isblocked at the promyelocyte stage (19, 20). The ability of adominant negative RARa to inhibit neutrophil differentiationat the promyelocyte stage was also reported in murine primarybone marrow cultures (20). The blocked promyelocytes pro-liferated continuously as a GM-CSF-dependent cell line butwere induced to terminally differentiate into neutrophils follow-ingRA treatment (20). These findings indicate that expression ofa dominant negative RARa in murine myeloid progenitors altersmyeloid maturation and may represent a transformation stepleading to leukemia. These data suggested that PML/RARaexpression in early myeloid cells would alter myelopoiesis.The present study was undertaken to explore the growth and

differentiation effects of PML/RARa in bone marrow my-eloid cells in the mouse. Mice were engineered to express afull-length PML/RARa cDNA transgene driven by the CD1 lbpromoter. PML/RARa expression in bone marrow cells wasconfirmed using a reverse transcription (RT) polymerase chainreaction assay. Clonal growth assays performed using periph-eral blood and bone marrow cells isolated from transgenicmice reveal PML/RARa expression leads to impaired myelo-poiesis. Unexpectedly, transgenic mice display marked sensi-

Abbreviations: APL, acute promyelocytic leukemia; PML, promyelo-cytic leukemia gene; RARa, retinoic acid receptor a; RA, all-trans-retinoic acid; GM-CSF, granulocyte/macrophage colony-stimulatingfactor; RT, reverse transcription; DMSO, dimethyl sulfoxide; HPP-CFC; high proliferative potential colony-forming cell; KL, kit ligand;CFU-GM, colony forming unit-granulocyte/macrophage; CFU-GEMM, mixed CFU; LPP-CFC, low proliferative potential colonyforming cell.1Present address: Department of Pharmacology, Kyoto UniversityFaculty of Medicine, Kyoto, Japan.ttTo whom reprint requests should be addressed at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

t*CNRS URA 1160 Laboratoire d'Oncologie Moleculaire, 1, RueCalmette, Institut Pasteur de Lille, France, 59019.

7900

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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tivity to sublethal radiation with increased depression of totalwhite blood cell (WBC) and granulocyte counts. The recoveryof granulocytes and leukocytes was enhanced by RA treatmentof transgenic mice. These findings are consistent with the viewthat PML/RARa inhibits myelopoiesis and directly contrib-utes to transformation of myeloid cells.

MATERIALS AND METHODSRT-PCR Assay and Southern Analysis. RNA and genomic

DNA were purified from NB4 cells and the bone marrow ofcontrol and transgenic PML/RARa mice using guanidinethiocyanate and standard techniques (21). PML/RARa ex-pression was assayed in total RNA isolated from harvestedbone marrow. Nested primers and an RT-PCR assay were usedwith established techniques optimized for detection of PML/RARa mRNA in APL cells (6, 17). Southern analysis wasperformed as described previously (22).PML/RARtx Transgenic Mice. The PML/RARa expres-

sion vector was constructed using a full-length PML/RARacDNA (4) cloned into the NotI site of a Bluescript (Stratagene)plasmid. This was gel-purified after digestion with BglII andXbaI. A HindIII/SmaI fragment of the CD11b promoter (29)was ligated at the HindlIl site of a Bluescript SK- plasmidcontaining a 0.7-kb XbaI/EcoRI fragment of the SV40Poly(A) sequence. The vector was engineered by bluntlyligating the 5' BglII site in the PML/RARa fragment to theSmaI site in the plasmid and ligating the 3' XbaI site of thisfragment to an XbaI site in the plasmid backbone. Transgenicmice were engineered using standard techniques (23). Foundermice were mated to ICR Swiss or non-Swiss mice. Controlswere identical to transgenic mice but did not express PML/RARa. An independent PML/RARa line was derived to ex-clude genetic heterogeneity as responsible for an observed phe-notype. Mice were aged 8-12 weeks and were sex and weightmatched. An approved institutional animal care and use com-mittee protocol was used.

Radiation Protocols. Control and transgenic mice receivedry irradiation from a cesium source (600 rad/mouse). Thiscaused negligible lethality, transient anemia, thrombocytope-nia, granulocytopenia, and minimal weight loss or othersymptoms in control mice. When radiation-induced leukope-nia developed, WBC and granulocyte counts, hemoglobin,hematocrit, and platelet counts were measured generally daily,until WBC recovery. Granulocytes were counted after crystalviolet staining of peripheral blood smears. To limit phlebot-omy, groups of six or 12 mice had base-line hematologicparameters measured. After radiation-induced leukopenia,three control or transgenic mice had hematologic parametersmeasured generally daily. Mice were rotated for these mea-surements. Results were correlated with survival after irradi-ation. Whether RA (Sigma) limited radiation-induced myelo-suppression in transgenic mice was studied. Twenty-fourPML/RARa transgenic mice were each irradiated with 600rad. The day before and of irradiation, 12 transgenic mice wereeach administered dimethyl sulfoxide (DMSO, Sigma) as a40-,ul i.p. injection. The day before and of irradiation, 12 othertransgenic mice were each administered RA (1 mg i.p.)dissolved in the same DMSO volume as controls.

Clonal Growth and Cell Sorter Assays. Bone marrow fromuntreated or 24-h post 5-fluorouracil treated mice were har-vested. Early hematopoietic progenitors were detected inprimary and 7-day "delta" culture generated-secondary highproliferative potential colony-forming cells (HPP-CFCs) usinginterleukin-3, KL, and established techniques (24-26). For5-fluorouracil-untreated mice, primary cultures were per-formed using peripheral blood or bone marrow with 5 x 104cells/ml supplemented with or without cytokines. Bone mar-row cultures were used for colony forming unit-granulocyte/macrophage (CFU-GM) and burst forming unit-erythroid

measurements. Low proliferative potential colony formingcells (LPP-CFCs) were assayed with 2.5 x 105 bone marrowcells. Cultures were scored at 7 days for CFU-GMs or LPP-CFCs. HPP-CFC colonies with minimum diameter of 0.5 mmwere scored at 10 days. Assays were performed in triplicate andconfirmed in at least triplicate independent experiments. Therecombinant cytokines were murine KL at 20 ng/ml (Immu-nex), murine GM-CSF at 10 ng/ml (Immunex), murine inter-leukin-3 (Intergen) at 20 ng/ml, human G-CSF (AmgenBiologicals) at 1000 units/ml, human M-CSF (Cetus) at 1000units/ml, and human erythropoietin (Amgen) at 2 units/ml.

Cell sorter assays compared CDllb+ populations in thebone marrow of these mice. Cells were incubated with a ratanti-mouse CD11b (MAC-1) fluorescein isothiocyanate-conjugated antibody or an isotype matched, fluorescein iso-thiocyanate-labeled, control. antibody (Caltag). Red bloodcells were lysed wtih potassium acetate. Assays were per-formed as described (17).

RESULTSTransgenic PML/RARa expression was confirmed using anRT-PCR assay (Fig. 1). The expected short form of PML/RARa mRNA (6) was found in bone marrow of transgenic(lane 6) but not control mice (lane 4). As a positive control,RNA was isolated from the APL cell line (NB4) that expressesthe long form of PML/RARa (6, 16) along with several splicevariants (Fig. 1 lane 2). Southern analysis was performed usinggenomic DNA from a representative transgenic mouse toconfirm the entire PML/RARa cDNA was present (data notshown). A full-length PML/RARa cDNA was cloned andsequenced from this mouse. No sequence additions, deletions,or substitutions were identified in this cDNA (data not shown).To begin to characterize the phenotype present in this

transgenic line, clonal growth assays were performed on bonemarrow and peripheral blood. As expressed per femur, inwild-type versus transgenic bone marrow, total burst formingunit-erythroid (4047 ± 3059 SEM versus 3814 ± 187 SEM)and CFU-GEMM (1017 ± 276 SEM versus 998 ± 276 SEM)numbers did not appreciably differ while CFU-GM numberswere slightly higher in wild-type mice (37,050 ± 21,739 SEMversus 29,386 + 566 SEM). In contrast, peripheral blood cellsderived from PML/RARa transgenic but not wild-type con-trols displayed a blunted clonal growth response to GM-CSF(Fig. 24, P value s 0.007, two-tailed Student's t test). Thiscytokine is active in the same myeloid populations in which theCD11b promoter is active (27-30). As expected, an inhibited

bp- 1000-- 800- 600- 500- 400- 300

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FIG. 1. The RT-PCR assay for PML/RARa mRNA expression inPML/RARa transgenic and control mice. To exclude contaminatinggenomic DNA as responsible for the PML/RARa signal, RNAsamples were treated with RNase-free DNAse. The lanes representtotal RNA from the NB4 cell line (lanes 1 and 2), pooled bone marrowfrom control mice (lanes 3 and 4), or pooled bone marrow fromPML/RARa transgenic mice (lanes 5 and 6). No RT was added tolanes 1, 3, and 5, while RT was added to the reactions of lanes 2, 4, and6. These species were size-fractionated on a 0.8% agarose gel, andmolecular weight markers are depicted in base pairs.

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FIG. 2. (A) Clonal growth assays performed on peripheral bloodcells derived from wild-type and PML/RARa transgenic mice. Thisassay reveals the normal stimulatory response of wild-type cells to thecytokine GM-CSF and an impaired response of transgenic cells.GM-CSF co-treatment with KL did not overcome this inhibition. Asa control, no cytokines were added. The results depict representativefindings from triplicate clonal growth assays conducted in five inde-pendent wild-type and five PML/RARa transgenic mice. Colonies per4 x 104 cells plated are reported with standard deviation barsindicated. (B) The cell sorter analysis for CD11b+ cells in wild-typeand PML/RARa transgenic bone marrow each isolated from repre-sentative mice. The control antibody is isotype matched, not recog-nizing CDllb. A similar cell number and fluorescence intensity isshown for CDllb+ populations in wild-type versus PML/RARatransgenic bone marrow.

clonal growth response was also observed after addition ofeither G-CSF or M-CSF (data not shown). GM-CSF co-treatment with KL did not overcome this inhibition (Fig. 2A,P value c 0.009, two-tailed Student's t test). Clonal growthassays were simultaneously performed with bone marrow fromfive control and five PML/RARa transgenic mice followingstimulation with GM-CSF or GM-CSF plus KL. When findingsare expressed per femur, bone marrow clonal growth was only

slightly higher in control versus transgenic mice followingtreatments with GM-CSF (10,555 ± 1377 SEM versus 9986 ±2226 SEM) or GM-CSF plus KL (11,505 ± 1595 SEM versus10,978 + 2105 SEM). Similar results were obtained from asecond transgenic line established from an independentfounder (data not shown).

It is unlikely the CD11b driven PML/RARa transgene isexpressed in the earliest myeloid progenitors (27-30). Stemcell assays (HPP-CFC) were performed with bone marrowharvested from PML/RARa transgenic or wild-type mice toconfirm that peripheral blood findings did not reflect observedchanges in myeloid stem cells. Indeed, the responses of theseearly myeloid progenitors to GM-CSF or GM-CSF plus KLwere normal. Thus, comparable LPP-CFC and HPP-CFCnumbers were isolated from 5 wild-type and 5 PML/RARatransgenic mice (data not shown). Similar findings were ob-tained in a second independent experiment, indicating thatchanges in stem cell., numbers do not account for alteredGM-CSF response in peripheral blood from the transgenicline. Similarly, search for changes in CDllb+ populationsusing cell sorter analysis failed to reveal significant differencesbetween control and transgenic mice (Fig. 2B). Similar find-ings were obtained in 22 wild-type and 20 PML/RARatransgenic mice. Thus, the numbers of CD11b+ bone marrowcells do not explain altered GM-CSF cellular response oftransgenic mice. To exclude other changes due to PML/RARaexpression, necropsies were performed in eight transgenicmice and were compared with findings obtained from eightcontrol mice. No gross abnormalities were observed in thesetransgenic or control mice. Histopathologic studies were per-formed on the spleen and bone marrow of mice. No abnor-malities were observed (data not shown).As shown in Fig. 2A, peripheral blood clonal growth assays

reveal an altered cytokine response ofPML/RARa expressingcells. To evaluate whether stressing the bone marrow of miceuncovers a phenotype, groups of wild-type and transgenic mice(matched for age, sex, and weight) were each irradiated withsublethal total body irradiation (600 rad, single dose). Micewere monitored at indicated time points for radiation-inducedchanges in hematologic parameters, including total WBC andgranulocyte counts. Results of a representative experimentappear in Fig. 3A. This demonstrates that, as compared withcontrols, PML/RARa transgenic mice have a more rapid andsustained decline in total WBC counts (Fig. 3A). The periodof severe granulocytopenia (

Proc. Natl. Acad. Sci. USA 93 (1996) 7903

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FIG. 3. (A) The percent WBC counts (PML/RARa transgenic versus wild-type mice) before and at indicated time points after a single doseof 600 rad/mouse. This figure demonstrates the transgenic mice had a more rapid and sustained suppression of WBC counts than controls. Thebars depict the standard error of the mean. (B) The survival curve of these irradiated wild-type versus PML/RARa transgenic mice. As expected,nearly all wild-type mice (94.4%) survived, while only (54.2%) PML/RARa transgenic mice survived irradiation. This difference is significant (Pvalues c 0.0043) as discussed in Results.

counts were 3-fold higher in RA-treated mwith controls (data not shown). Results wduplicate independent experiment. Thus, iPML/RARa transgenic mice antagonizes rgranulocytopenia.

DISCUSSIONThis study reports that CD11b driven PML/Iexpression in mice impairs myelopoiesis inCDllb is the a chain of the Mac-1 integrin an

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[ice as compared expressed in myeloid cells (27-30). The CD11b promoterrere similar in a directs cell type-specific expression in myelomonocytic cells,{A treatment of which are known to be sensitive to GM-CSF (27-30). Since itadiation induced is likely that the CD11b promoter is not active in myeloid

progenitors (27-30), it is expected that changes in HPP-CFCand LPP-CFC progenitors would not be found. In contrast,PML/RARa transgenic expression causes marked suppres-sion of CFU-GMs in peripheral blood clonal growth assays, as

ZARa transgenic shown in Fig. 2A. The in vitro suppression of CFU-GMs is notvivo and in vitro. overcome when PML/RARa myeloid cells are cultured withd is preferentially GM-CSF and KL. Those colonies forming from control and

transgenic peripheral blood were of a smaller size (50-200cells/colony) than CFU-GMs in bone marrow (50-10,000cells/colony) and probably represent a more differentiatedprogenitor population. Notably, a minimal suppression ofCFU-GMs is present in clonal growth assays performed withbone marrow derived from the PML/RARa transgenic line.

* This difference between clonal growth results from peripheral,°10 blood versus bone marrow cells could reflect the reduced

capacity of the PML/RARa transgene to mobilize progenitormyeloid cells to peripheral or extramedullary sites. This may

JI'd partly account for the enhanced sensitivity of these transgenic, mice to irradiation. This finding is not without precedence. For

example, mice bearing mutant genes at theW or Steel loci have4 \ modest changes observed in bone marrow clonal growth

assays, yet these mice also have, as compared with wild-typemice, enhanced radiation sensitivity (32-34).

* Although almost all control mice survived sublethal irradi-ation, nearly 50% of PML/RARa mice succumbed (Fig. 3B).This lethality was associated with a greater degree of granu-locytopenia and leukopenia (Fig. 3A) in PML/RARa versuscontrol mice. Other cellular defects in addition to granulocy-topenia and leukopenia could contribute to this increasedlethality. This includes altered granulocytic functions (migra-tion and phagocytosis) and altered stromal-bone marrow

67 8interactions or splenic extramedullary hematopoiesis (35).

6 7 8 Although multiple factors may contribute to this lethality, it isapparent that RA treatment antagonizes some inhibitoryeffects of PML/RARa on myelopoiesis. RA treatment of

PML/RARa mice these transgenic mice enhances granulocyte recovery (Fig. 4).-ment with DMSO Finding an inhibitory effect of PML/RARa expression onstudy demonstrates myelopoiesis is not unexpected. Prior work reveals that in therecovery following absence of ligand, PML/RARa functions as an inhibitorynic mice. The more transcription factor with putative dominant negative functionsignificant (depicted (4, 5, 11, 12). In certain myeloid leukemic cells, engineered

t.TheySaxisndepicts PML/RARa overexpression inhibits induced differentiationint, and the x axis (11, 15). In defined cell contexts, PML/RARa expressionline indicates that exerts an inhibitory effect on the transactivation of reporters5. containing retinoic acid response elements (4, 5, 11, 12). Also,

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PML/RARa expression leads to aberrant nuclear localizationof PML in APL cells (13, 14), and RA restores a normalnuclear localization pattern. Overexpression of either PML(17, 18) or RARa (17) in NB4 cells leads to growth suppres-sion either through a direct growth suppressive effect of PMLor RARa or indirectly through an antagonism of the inhibitoryPML/RARa effects in APL cells (17).The inhibition of myelopoiesis reported here has clinical

implications. That RA treatment of irradiated mice antago-nizes the granulocytopenia found in PML/RARa transgenicmice is reminiscent of the clinical retinoid response of APLpatients. RA treatment overcomes the clinical maturationblock found in leukemic promyelocytes, presumably due toantagonism of the actions of PML/RARa in these cells. SinceDMSO protects against radiation damage (31), this likelyaccounts for the more limited granulocytopenia found invehicle-treated and irradiated PML/RARa mice (Fig. 4) thanin transgenic or control mice not treated with DMSO (Fig. 3and data not shown).Of note, these PML/RARa transgenic mice do not develop

leukemia or obvious hematopathology, despite more than 2years of follow-up. Several factors could contribute to thesefindings. The low levels of PML/RARa expressed in bonemarrow cells from these transgenic mice suggest that anoptimal PML/RARa gene dosage is required to fully trans-form myeloid cells. Appropriate timing of PML/RARa ex-pression in early myeloid cells might play a critical role intransformation. Perhaps a myeloid-specific promoter active ineven earlier myeloid cells than the CD11b promoter used toengineer this transgenic line will yield higher levels of PML/RARa expression in these progenitors. Alternatively, a secondoncogenic event such as ras or p53 activation may be requiredto develop leukemia (36,37). This PML/RARa transgenic linemay prove useful to identify second oncogenic events contrib-uting to APL development. Since RA treatment of thisirradiated PML/RARa line antagonizes some of the inhibi-tory PML/RARa effects, this transgene might predict usefultherapeutic properties ofRA or retinoid analogs before use inAPL clinical trials.

In summary, these studies provide the first direct evidencethat expression of PML/RARa in mice can trigger abnormalmyelopoiesis and increased radiation sensitivity. RA therapyreverses at least some of these findings, suggesting that PML/RARa mice provide a useful model to further understand thebasis of oncogenic transformation and treatment of APLpatients.

We thank Janet Allopenna and Harry Satterwhite for experttechnical assistance. We thank Yue Tao, Department of Biostatistics,Memorial Sloan-Kettering Cancer Center, for helpful consultations;Dr. M. Lanotte, INSERM, France for the gift of the NB4 cell line; andDr. D. Tenen, Harvard Medical School, for the gift of the CD11bpromoter. R.M.E. is an Investigator of the Howard Hughes MedicalInstitute for Biological Studies. These studies were supported byNational Institutes of Health (NIH) Grant R01-62275-02 to E.D., theHoward Hughes Medical Institute for Biological Studies, and NIHProgram Project CA55418 to R.M.E. K.N-B. was supported by NIHNational Research Service Award lF32CA61646-OlA1.

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