Dok1 and Dok2 Proteins Regulate Cell Cycle in Hematopoietic Stem and Progenitor Cells Emilie Coppin, Maria De Grandis, Pier Paolo Pandolfi, Marie-Laure Arcangeli, Michel Aurrand-Lions and Jacques This information is current as A. Nunès of September 26, 2021. J Immunol published online 18 April 2016 http://www.jimmunol.org/content/early/2016/04/16/jimmun ol.1501037 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 18, 2016, doi:10.4049/jimmunol.1501037 The Journal of Immunology

Dok1 and Dok2 Proteins Regulate Cell Cycle in Hematopoietic Stem and Progenitor Cells

Emilie Coppin,*,†,‡,x Maria De Grandis,*,†,‡,x Pier Paolo Pandolfi,{,‖,# Marie-Laure Arcangeli,*,†,‡,x,1 Michel Aurrand-Lions,*,†,‡,x,2 and Jacques A. Nune`s*,†,‡,x,2

Dok1 and Dok2 proteins play a crucial role in myeloid cell proliferation as demonstrated by Dok1 and Dok2 gene inactivation, which induces a myeloproliferative disease in aging mice. In this study, we show that Dok1/Dok2 deficiency affects myeloprolif- eration even at a young age. An increase in the cellularity of multipotent progenitors is observed in young Dok1/Dok2-deficient mice. This is associated with an increase in the cells undergoing cell cycle, which is restricted to myeloid committed progenitors. Furthermore, cellular stress triggered by 5-fluorouracil (5-FU) treatment potentiates the effects of the loss of Dok proteins on multipotent progenitor cell cycle. In addition, Dok1/Dok2 deficiency induces resistance to 5-FU–induced hematopoietic stem cell

exhaustion. Taken together, these results demonstrate that Dok1 and Dok2 proteins are involved in the control of hematopoietic Downloaded from stem cell cycle regulation. The Journal of Immunology, 2016, 196: 000–000.

yeloid cells are sensitive to cytokines and growth in hematopoietic cell transformation as highlighted in chronic factors that activate intracellular protein tyrosine myeloid leukemia with the BCR-ABL fusion gene product showing M kinases (PTK). Constitutive PTK activation results PTK activity. Among the main substrates identified downstream

BCR-ABL, the adaptor proteins, downstream of kinase Dok1 and http://www.jimmunol.org/ Dok2 (1, 2), are RasGAP-binding proteins acting as negative *INSERM, U1068, Centre de Recherche en Cance´rologie de Marseille, 13009 Mar- regulators of signaling pathways in lymphoid (3, 4) and myeloid seille, France; †Institut Paoli-Calmettes, 13009 Marseille, France; ‡Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 7258, Centre de Recherche en cells (1, 2, 5, 6). Dok1 and Dok2 proteins have been shown to x Cance´rologie de Marseille, 13009 Marseille, France; Universite´ d’Aix-Marseille, inhibit growth factors and cytokine-induced cell proliferation in UM105, 13009 Marseille, France; {Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Harvard Medical School, Boston, MA 02215; ‖Department of Med- hematopoiesis (6–11). Mice deficient for both Dok1 and Dok2 icine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA double knockout (Dok DKO) develop a myeloproliferative disease # 02215; and Department of Pathology, Beth Israel Deaconess Medical Center, Harvard similar to human chronic myelomonocytic leukemia (CMML) Medical School, Boston, MA 02215 DOK2 1 with a latency of 1 y. We recently identified loss-of-function

Current address: Inserm U967, Institut de Recherche en Radiobiologie Cellulaire et by guest on September 26, 2021 Mole´culaire – Commissariat a` l’E´ nergie Atomique et aux E´ nergies Alternatives, point mutations in CMML patients (12). Dok DKO mice display Fontenay-aux-Roses, France. medullary and extramedullary hyperplasia and increased activa- 2M.A.-L. and J.A.N. contributed equally to this paper. tion of RAS and PI3K-dependent pathways in cytokine-activated ORCIDs: 0000-0001-6465-8723 (M.D.G.); 0000-0002-8361-3034 (M.A.-L.). granulomonocytic progenitors (6, 7). Received for publication May 6, 2015. Accepted for publication March 11, 2016. In healthy adult mammals, early hematopoietic precursors are mainly located in the bone marrow (BM). They are part of the Lin2 This work was supported by institutional grants from INSERM, Centre National de la + + Recherche Scientifique and Universite´ d’Aix-Marseille Centre de Recherche en Can- Sca-1 Kit (LSK) compartment that can be further divided into ce´rologie de Marseille, Fondation pour la Recherche Me´dicale (Equipe Fondation hematopoietic stem cells (HSC) and multipotent progenitors pour la Recherche Me´dicale Grant DEQ20140329534), Immunity and Cancer team (to J.A.N.), and by specific grants from Fondation Association pour la Recherche (MPP) 1–4 using additional markers such as CD34, CD150, sur le Cancer (Grant PJA20141201656 [to J.A.N.] and Grant PJA20141201990 [to CD48, and CD135 (13–16). HSC represent a very small cell M.A.-L.]) and Sites de Recherche Inte´gre´e sur le Cancer (INCa-DGOS-Inserm Grant population with a clonal capacity to provide lifelong regeneration 6038 [to M.A.-L.]). E.C. was supported by fellowships from the Re´gion Provence Alpes Coˆte d’Azur–Innate Pharma and Fondation Association pour la Recherche sur of all differentiated hematopoietic cells. To maintain hematopoi- le Cancer. M.D.G. was supported by a fellowship from Fondation de France and etic homeostasis, HSC differentiation, self-renewal, and mainte- M.-L.A. by a fellowship from the Fondation pour la Recherche Me´dicale. nance must be tightly regulated (17–20). Most adult HSC are E.C. designed the experiments, wrote the paper, and performed most of the experi- quiescent, contributing to HSC maintenance in the BM and en- ments and data analysis; M.D.G. performed some experiments and analyzed data; P.P.P. generated deficient mice; and M.-L.A., M.A.-L., and J.A.N. designed the experiments suring lifelong hematopoietic replenishment (19, 21). Further- and wrote the paper. more, slow cycle progression is essential to maintain all lifelong Address correspondence and reprint requests to Dr. Emilie Coppin at the current address: pools of self-renewing HSC (22). To avoid exhaustion, HSC must Heart and Vascular Institute, Division of Cardiology, Department of Medicine, University be protected from stress. Indeed, quiescent HSC populations are of Pittsburgh Medical Center, Pittsburgh, PA 15213, or Dr. Jacques A. Nune`s, Centre de Recherche en Cance´rologie de Marseille, CS 30059, 27 Boulevard Lei Roure, 13273 resistant to 5-fluorouracil (5-FU)–induced myelosuppression (23, Marseille Cedex 9, France. E-mail addresses: [email protected] (E.C.) or jacques. 24), suggesting that HSC quiescence is closely linked to the [email protected] (J.A.N.) protection of the hematopoietic system from various stresses. The online version of this article contains supplemental material. Several mechanisms regulate HSC cell cycle and maintain cell Abbreviations used in this article: BM, bone marrow; CDK, cyclin-dependent kinase; quiescence (24–27). Tie2/Angiopoietin-1, Mpl/thrombopoietin CMML, chronic myelomonocytic leukemia; CMP, common myeloid progenitor; b DKO, double knockout; 5-FU, 5-fluorouracil; GMP, granulocyte monocyte progen- (TPO), and Wnt/ -catenin signaling pathways; cell adhesion itor; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; molecules; and p21 and p57 Cyclin-dependent kinase (CDK) in- 2 + 2 2 + + LK, Lin Kit Sca-1 ; LSK, Lin Sca-1 Kit ; MPP, multipotent progenitor; PTK, hibitors are involved in the maintenance of HSC and protect them protein tyrosine kinase; TPO, thrombopoietin; WT, wild-type. from cellular stress (25–31). Because Dok proteins are critical Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 negative regulators of proliferation and consequently of cell cycle

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501037 2 Dok1 AND Dok2 REGULATE HSPC CELL CYCLE downstream of growth factor signaling, we wondered whether Lineage depletion they could play a role in HSC quiescence. BM cells were incubated in PBS plus 2% FBS and 2.5 mM EDTA with To test this hypothesis, we analyzed the effects of double Dok1 biotinylated Abs against lineage markers, followed by anti-biotin and Dok2 gene deletion on hematopoietic precursors in young microbeads (Miltenyi Biotec) and then the Lineage+ cells were depleted mice. We show imbalanced frequencies and numbers of mature by autoMACS Pro Separator (Miltenyi Biotec). hematopoietic cells and precursors at steady state with an in- Cell cycle analysis creased proportion of cells in active phases of cell cycle. Inter- estingly, upon 5-FU–induced myeloablation, Dok DKO HSC BM progenitors were enriched as previously described and stained with c-Kit-allophycocyanin-eFluor750, CD150-allophycocyanin, Sca-1-PerCP- return to a quiescent state faster than wild-type (WT) HSC. Serial Cy5.5, CD48-PECy7, and CD16/32-PECy7. The biotin-conjugated line- 5-FU injections of these mice demonstrated that Dok DKO HSC age combination was revealed with Alexa Fluor 594-streptavidin (BD show enhanced resistance to exhaustion compared with WT mice. Biosciences). After extracellular staining, cells were permeabilized, fixed, Taken together, our results reveal a new unexpected function of and stained with anti–Ki67-FITC Ab (BD Biosciences) (32) and DAPI (Sigma-Aldrich) as described previously (33). Dok proteins as switch controllers of cell cycle entry, promoting quiescence at steady state and increasing susceptibility to HSC Gene expression analysis in hematopoietic stem and progenitor exhaustion upon myeloablative stress. cells Ten cells for each population (HSC, MPP1, MPP2, and MPP3/4) from WT Materials and Methods or Dok DKO mice were sorted using the autoclone module on an Aria III Mice SORP sorter (BD Biosciences) into 96-well plates in the RT-STA Master

Mix Solution. Cell lysis, cDNA synthesis, and amplification were per- Downloaded from Dok-12/2Dok-22/2 DKO mice (Dok DKO) were obtained by interbreeding formed according to Fluidigm protocols. Selected TaqMan Gene Ex- Dok-12/2 mice with Dok-22/2 mice both in 129/Sv background as de- pression assays (original magnification 320) were pooled and diluted scribed previously (2, 7). We generated CD45.1+ and CD45.2+ mice for with water 100-fold so that each assay is at a final concentration of 0.23 chimera and transplantation experiments by crossing a 129/Sv (CD45.2+) in the pooled assay mix. Five microliters of CellsDirect 23 Reaction + male and CD45.1 C57BL/6 female to obtain F1(129/Sv 3 C57BL/6) Mix (Invitrogen), 2.5 ml pooled assay mix, 0.2 mlSuperScriptIIIRT/ CD45.1+/CD45.2+ recipient mice. WT 129/Sv and CD45.1+ C57BL/6 Platinum Taqmax mix, and 2.3 ml water were combined to a final volume mouse strains were purchased from Charles River Laboratories of 10 ml in 1 well of a 96-well qPCR plate for the sort. cDNA samples (L’Arbresle, France). Six- to eight-week-old females were used throughout were amplified using the following program (1): 50˚C, 10 min (2); 95˚C, http://www.jimmunol.org/ the study. All experiments were performed in agreement with the French 2 min (3); 95˚C, 15 s (4); 60˚C, 4 min; repeat steps (3) and (4) 18 times. guidelines for animal handling and were approved by the Inserm ethical Preamplified products were diluted 5-fold before analysis with Universal committee. PCR Master Mix and inventoried TaqMan gene expression assays in 96.96 Dynamic Arrays on a BioMark System (Fluidigm). Nomenclature BM transplantation assays of the used TaqMan probes and targets is reported in Supplemental Table I. For each gene, the relative expression values were calculated Freshly dissected femurs, tibias, and hips were isolated from mice. BM was as 40 – cycle threshold. flushed with a syringe into RPMI 1640 medium supplemented with 10% FBS. The BM was spun at 1600 rpm by centrifugation at 4˚C, and RBCs HSC exhaustion after 5-FU treatment were lysed in ACK Lysing Buffer (Life Technologies) for 3 min. After centrifugation, cells were suspended in PBS plus 2% FBS, passed through In survival experiments, Dok DKO mice and control mice were injected by guest on September 26, 2021 a cell strainer (BD Biosciences), and counted. For serial transplantation, i.p. once per week with 5-FU (120 mg/kg). Survival was monitored on 5 3 106 total BM cells of WT or Dok DKO CD45.2+ were transplanted via a daily basis up to 25 d after the first 5-FU injection. In others ex- retro-orbital injection into lethally irradiated (9.5Gy) F1(129/Sv 3 C57BL/6) periments, animals received just two 5-FU injections (days 0 and 21, CD45.1+/CD45.2+ mice. This model was chosen to keep an allelic reporter when peripheral blood leukocyte count returns to a similar level than at system and to overcome graft rejection because Dok DKO cells were on day 0). 129/Sv background (H-2bc) different from the MHC background of C57BL/6 mice (H-2b) (for details see (http://www.imgt.org/IMGTrepertoireMHC/ Hematopoietic colony-forming cell assay Polymorphism/haplotypes/mouse/MHC/Mu_haplotypes.html#polymorphism). Fresh GMP or BM cells treated with ACK Lysing Buffer (Life tech- Mouse chimerism in the peripheral blood was monitored by flow cytometry nologies) were counted and diluted to a concentration of 5 3 105 cells/ml every 4 wk (weeks 4, 8, and 12). Chimerism and hematopoietic phenotype in in IMDM supplemented to 10% FBS (Eurobio), 100 U/ml penicillin, and the BM and spleen were evaluated at 12 wk after transplantation. 100 mg/ml streptomycin (Life Technologies). For comparison of GMP Flow cytometry and cell sorting differentiation potential between Dok DKO and WT (Fig. 3), 200 sorted cells were added to 2 ml Methocult GF M3434 (Stemcell Technologies) The following Abs used for flow cytometry and cell sorting were pur- and seeded at 1 ml/dish. For total BM CFU-GM assays, 2 3 104 cells and chased from eBioscience or BioLegend: biotinylated anti-CD4 (clone Methocult GF M3534 were used. After 7–10 d, the dishes were scored RM4-5), CD8 (clone 53-6.7), CD3 (clone 2C11), DX5 (clone DX5), for hematopoietic colonies, and results were expressed as number of CD11c (clone N418), CD19 (clone 6D5), B220 (clone RA3-6B2), colonies per well. TER119 (clone TER119), CD11b (clone M1/70) and Gr1 (clone RB6- 8C5) (Lineage mixture), cKit-allophycocyanin-eFluor780 (clone 2B8), Immunohistochemistry Sca-1-PerCP-Cy5.5 (clone D7), CD34-FITC (clone RAM34), CD16/32- Bones were decalcified during 15 d in PBS plus 270 mM EDTA solution at PECy7 (clone 93), CD150-allophycocyanin (clone TC15- 12F12.2), 37˚C and then fixed in 1% paraformaldehyde and prepared to paraffin CD48-PECy7 (clone HM48.1), CD48-BV421 (clone HM48.1), CD135- inclusion using automated tissue processor ASP 300 (Leica). Dehydra- PE (clone A2F10), CD45-allophycocyanin-eFluor780 (clone 30-F11), tion, clarification, and infiltration steps were performed by successive CD45.1- PE CF594 or -FITC (clone A20), CD45.2-AF700 or -eFluor 450 absolute ethanol, histolemon, and paraffin baths. Then bones were dis- (clone 104), CD19-AF700, CD19-allophycocyanin-Cy7, CD11b-FITC, posed in paraffin. Sections of 5-mm thickness were performed with HM CD3-PECy7, CD115-allophycocyanin (clone AFS98), F4/80-PE (clone 340E microtome (Thermo Scientific). Hematoxylin eosin safran staining BM8), Ly6C-BV421 (clone AL-2), Ly6G-BV711 (clone 1A8), Ly6G- was performed using automated slide stainer JUNG XL (Leica). Finally, BV421, CD71-PE (clone PE C2), and Ter119-BV421 and CD105-PE slides were dehydrated by absolute ethanol and histolemon baths and (clone MJ7/18). Brilliant Violet 510-Streptavidin (BioLegend) and AF594- mounted in Pertex medium using glass coverslipper CV5030 (Leica). Streptavidin (BD Biosciences) were used. For CFU assays, granulocyte Slides were scanned with a digital slide scanner NanoZoomer 2.0-HT monocyte progenitor (GMP) cells were obtained after total BM cells sorting 2 2 (Hamamatsu). according to their surface markers expression (Lin c-Kit+Sca-1 CD34+ and + CD16/32 ). Flow cytometry was performed on a BD LSR II flow cytometer, Statistical analysis and cell sorting was executed on a BD FACSAria III or BD FACSAria II SORP. Flow Cytometry data were analyzed with BD FACSDiva version Results are expressed as mean values 6 SEM. Statistical significance of 6.1.2 software (BD Biosciences). differences between the results was assessed using a two-tailed unpaired The Journal of Immunology 3

Student t test, performed using GraphPad Prism version 5.03 software indicating a strong hematopoiesis reconstitution (Fig. 2A, (GraphPad). The p values , 0.05 were considered statistically significant. Supplemental Fig. 2B). Mice transplanted with Dok DKO BM showed the same myeloproliferative features than Dok DKO Results mice: monocytosis, splenomegaly, BM hypercellularity, in- Dok1 and Dok2 control cellularity in hematopoietic organs creased level of LK (Lin2Kit2Sca-12 cells), and LSK com- Single Dok1-orDok2-deficient mice did not show obvious partment beginning from MPP2 differentiation step (Fig. 2B, phenotypes possibly because of overlapping functions of Dok1 Supplemental Fig. 2C, 2D). These results demonstrate that the and its closest family member, Dok2 (34). In contrast, double hematologic disorders observed in Dok DKO mice are trans- deficiency for Dok1 and Dok2 results in myeloproliferative plantable and cell autonomous. In addition, serial transplanta- disorder resembling CMML after a latency of 1 yr (6, 7). Thus, tions showed an amplification of the observed phenotype, we analyzed hematopoiesis in 6- to 8-wk-old double Dok1 and suggesting that Dok DKO hematopoietic cells present an early Dok2 deficient (Dok DKO) mice to better understand the mo- commitment defect evolving with time (Fig. 2D, Supplemental lecular mechanisms controlled by Dok1 and Dok2 in hemato- Fig. 2C, 2D). Frequencies of LK and LSK cells were similar in poietic stem and progenitor cells (HSPC). Although absolute WT and mutant mice at steady state but significantly increased numbers of WBC were similar in WT and Dok DKO mice in the after serial transplantations (Fig. 2E). In secondary transplants, peripheral blood, a significant increase of monocytes (CD11b+ CMP are increased compared with the first transplant or steady Gr1loF4/80+) and neutrophils (CD11b+Gr1hi) was observed in state (Fig. 2D). Although first and secondary transplanted mice Dok DKO mice as compared with WT (Fig. 1A). The cellularity have similar frequency of donor cells in the peripheral blood,

+ Downloaded from of BM and spleen (data not shown) was significantly higher in tertiary Dok DKO BM–transplanted mice present increased CD45.2 Dok DKO than WT mice with significant increase in neutrophils donor cells frequency compared with WT BM transplanted mice + and monocytes, respectively, CD11b+Ly6GHigh/Ly6CLow and (respectively 87.95 6 1.05 versus 82.71 6 1.65% of CD45.2 Ly6GNeg/Ly6CHigh in the BM (Fig. 1B) and peripheral blood cells) (Fig. 2A, 2C). This suggests that Dok DKO HSC have (data not shown). Thus, Dok DKO mice already display fea- higher engraftment capacity. Indeed, whereas Dok DKO HSC and tures of hematopoietic disorders at a young age, suggesting a MPP1 are less abundant in the LSK cell compartment than the constitutive imbalanced commitment of hematopoietic progen- WT cells at steady state, after three rounds of transplantation their http://www.jimmunol.org/ itors. This was confirmed by the analysis of early hematopoi- proportion in the LSK cell compartment is higher (Fig. 2F, 2G), etic progenitors as defined by the gating strategy described in suggesting that Dok1 and Dok2 play a role as modifiers of HSPC Fig. 1C. A significant increase of cell numbers in Dok DKO expansion. compared with WT mice was detected in the Lin2Kit+Sca-12 Dok DKO HSC are endowed with higher engraftment and (LK) and LSK compartments. In different LSK subsets, the transient myeloid amplification capacities absolute number of HSC (CD150+,CD342,CD482,CD1352) and MPP1 (CD150+,CD34+,CD482,CD1352)remainedun- To address the question whether the increased numbers of myeloid changed, but their frequency was decreased (Fig. 1D, 1E). cells in Dok DKO mice (Fig. 1F) was due to increased prolifer- However, a significant increase in the abundance and frequency ation or differentiation of GMP into monocytes/macrophages, by guest on September 26, 2021 of the more differentiated subsets (MPP2 [CD150+,CD48+, CFU assays were performed using equal numbers of GMPs iso- CD1352], MPP3 [CD1502,CD48+,CD1352], and MPP4 lated from Dok DKO or control mice. Increased numbers of col- [CD1502,CD48+,CD135+]) was observed in Dok DKO com- onies with enlarged appearance were obtained using GMPs pared with control mice. In the LK cell compartment, the fre- isolated from Dok DKO as compared with control (Fig. 3A, 3B). quency of GMP was significantly increased at the expense of This was likely because GMP isolated from Dok DKO mice were common myeloid progenitors (CMP), whereas differences in less quiescent as compared with control (data not shown). Char- absolute numbers were only significant for GMPs, with an in- acterization of CFU-GM colonies showed increased percentage of crease because of DKO BM hypercellularity (Fig. 1F). How- Dok DKO cells expressing myeloid differentiation markers such ever, the ratio of CD1052CD1502 and CD1052CD150+ CMP as CD11b, CD115, or CD16/32 (Fig. 3C, 3D) with higher fre- subtypes (35) remained unchanged (Supplemental Fig. 2A, quencies of cells with a more differentiated aspect (Fig. 3E). 2B). Finally, an increase in basophilic and orthochromato- These results indicate that GMPs isolated from Dok DKO have an philic erythroblasts (36) was observed in Dok DKO mouse BM increased proliferation rate and differentiation potential into more (Supplemental Fig. 2C, 2D). Altogether, these data show that mature myeloid cells. Dok proteins are involved in hematopoiesis from early stages Dok1 and Dok2 control hematopoietic progenitor quiescence of hematopoietic differentiation with downstream consequences on the balanced proportions of CMP, GMP, and erythroid Because the size of the HSC pool is tightly controlled by the progenitors. balance between dormant and cycling cells, we further investi- gated the cell cycle status of the LK and LSK cell compartments Features of Dok DKO hematopoietic cells are transplantable in Dok DKO mice. We combined cell surface staining to define and cell autonomous hematopoietic subsets with intracellular staining with DAPI Because Dok1 and Dok2 expression is not restricted to hema- (DNA staining) and anti-Ki67 Ab (a cell proliferation associated topoietic cells, we tested whether hematopoietic defects are cell marker). We found less quiescent LSK and LK cells in Dok DKO autonomous. Lethally irradiated recipient mice (F1(129/Sv 3 mice than in WT control mice (Fig. 4A, 4B). In the LK cell + + C57BL/6) CD45.1 /CD45.2 ) transplanted with WT or Dok compartment, a decrease in the proportion of G0-phase cells with + DKO total BM (129/Sv CD45.2 ) were sacrificed 12 wk after an accumulation in G1 phase was detectable for CMP and GMP transplantation. The percentage of chimerism was monitored in but not for megakariocytic erythroid progenitor subsets (data not the peripheral blood of recipient mice, and similar reconstitution shown). In addition, analysis of LSK subsets revealed that the potential was observed between WT and Dok DKO BM donors cell cycle was affected in MPP1, MPP2, and MPP3/4 subsets but (Fig. 2A). Hematopoietic donor cells (CD45.2+)chimerism not HSC at the steady state (Fig. 4C), suggesting that Dok1 and represents up to 95% of the peripheral blood, spleen, and BM, Dok2 control cell cycle entry at the transition between HSC and 4 Dok1 AND Dok2 REGULATE HSPC CELL CYCLE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Dok1 and Dok2 proteins control cell numbers in hematopoietic organs at steady state. Absolute number of cells in peripheral blood (A) and BM (B) of indicated mice. Cell counts in peripheral blood were obtained using a dedicated hematology analyzer, and BM cell counting was achieved using flow cytometry. (C) FACS gating strategy of progenitors. Cells are gated from lineage-negative cells. (D) Absolute numbers of Lin2, LK, and LSK cells in BM. (E) Percentages and absolute numbers of HSC and MPP subsets within LSK compartment; (F) percentages and absolute numbers of megakariocytic erythroid progenitor (MEP), CMP, and GMP within the LK compartment. n, Dok DKO mice; N, WT animals. Mice are 6-8 wk old. Representative results of at least three independent experiments with n =6.*p , 0.05, **p , 0.01.

MPP1. To get further insights into the mechanism by which loss from Dok DKO or control mice. Surprisingly, we found a number of Dok1/Dok2 expression promotes cell cycle in MPPs, we of downregulated genes in the HSC compartment although we compared gene expression of cell cycle regulators such as Rb1 failed to detect Dok1 and Dok2 expression in HSC (Fig. 4D). and Rbl1 (negative regulators of cell cycle), cyclins (Ccns), This is likely because Dok1 and Dok2 are only expressed by a cyclin-dependent kinases (Cdks), cyclin-dependent kinase in- fraction of HSC and are thus not detected under our experimental hibitors (Cdkns), growth factor receptor (Mpl, Csf2ra,andKit), conditions (bulk of 10 sorted cells). In addition, we observed and transcription factors (Gata3 and Myb) between cells isolated robust upregulation of Cdkn1a and strong downregulation of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Serial transplantations reveal cell autonomous properties of Dok DKO HSPC. Percentage of chimerism observed in peripheral blood of lethally irradiated mice 12 wk after engraftment with WT or Dok DKO BM in primary (A), secondary, and tertiary transplants (C). p = 0.0232 for third transplantation. (B– D) Absolute numbers of LK and LSK subsets after primary or secondary transplants are shown. (E) Increased proportions of Lin2 cells,LSK,LKcompartments in Dok DKO–transplanted mice as compared with controls after serial transplantations. (F) Increased proportions of HSC and MPP1 compartments in Dok DKO transplanted mice as compared with controls after tertiary transplantations. (G) Representative example of increase for Dok DKO HSC+MPP1 subset (CD150+, CD482) proportions in BM LSK–gated cells between steady state and third transplantation. n = 10 for each genotype. *p , 0.05, **p , 0.01, ***p , 0.001.

Csf2ra expression in MPPs isolated from Dok DKO mice as occur in more mature progenitors, although the results pinpoint compared to control. This indicates that a dynamic complex to a prominent function of Dok1 and Dok2 in controlling HSC interplay between Dok signaling and cell cycle regulation may proliferation at the transition to MPPs. 6 Dok1 AND Dok2 REGULATE HSPC CELL CYCLE Downloaded from http://www.jimmunol.org/

FIGURE 3. Increased myeloid differentiation potential of GMP isolated from Dok DKO as compared with control. (A) Number of granulocytic- monocytic CFUs starting from 200 sorted GMPs from WT or Dok DKO mice. (B) Aspect of the colonies of the indicated genotype (original magnification 310). (C) Representative flow cytometry dot plots of cells recovered from CFU-GM methyl cellulose assay after 10 d. (D) Percentages of cells with the by guest on September 26, 2021 indicated phenotype. **p , 0.01, one representative experiment out of two; n = 6 mice/group. (E) Representative pictures of cytospin preparation of cells recovered from CFU-GM methyl cellulose assay after 10 d (original magnification 340).

Dok1 and Dok2 proteins control the cell cycle entry and difference was even more pronounced with BM cellularity in Dok myeloid cell cellularity after 5-FU injections DKO mice being twice as much as compared to WT mice, with To alter steady-state hematopoietic homeostasis and to induce increased progenitors with a granulomonocytic fate (Fig. 6B, left peripheral blood replenishment from proliferating progenitors, we and middle panels). This elevated BM cellularity in Dok DKO treated mice with 5-FU on days 0 and 21 and monitored leukocyte mice compared with WT littermates has been confirmed by per- counts in peripheral blood over 42 d. We found that WBC counts forming immunohistochemistry experiments (Supplemental Fig. were higher in the recovery period in Dok DKO mice as compared 3A). In the LSK cell compartment, the number of MPP1 to to WT mice (9.14 6 0.80.103 versus 6.03 6 0.47.103/mm3, re- MPP4 cells was higher in Dok DKO mice as compared to WT spectively, at day 14; Fig. 5A). This difference was enhanced littermates, whereas HSC numbers were not affected (Fig. 6B, during the second round of 5-FU injection. 5-FU stress-induced de right panel). Because HSC proliferation is tightly associated with novo hematopoiesis significantly increased the myelomonocytic their differentiation, one possibility is that Dok1 and Dok2 pro- phenotype because Dok DKO mice displayed obvious neutrophilia teins control HSC cell cycle entry, which is revealed by increased and monocytosis at day 14 (Fig. 5B). A selective increase of numbers of more differentiated progenitors. This was confirmed Ki67-positive myeloid cells was observed in Dok DKO leuko- by analysis of the cell cycle status of HSC and MPP1 at days 3 and cytes during the leukocyte proliferative phase (from days 3 to 9) 5 after 5-FU injection (Supplemental Fig. 3B). Indeed, in Dok (Fig. 5C), suggesting that loss of Dok protein expression promotes DKO samples, fewer HSC were quiescent (G0 cell cycle phase) cell cycle entry and proliferation in mature myeloid cells. than in WT samples at day 3 (Fig. 6C), but they returned to quiescence at day 5 in greater proportion (Fig. 6D), suggesting Dok1 and Dok2 proteins control cell cycle entry of HSC after that Dok1 and Dok2 regulate cell cycle checkpoints. This directly 5-FU–induced myeloablation affects progenitor cell numbers and differentiation because cell To confirm that our previous observations were due to deregula- cycle entry is known to occur at the transition between HSC and tions in cell cycle checkpoints in more immature progenitors, 6- to MPP1. This was confirmed by paired analysis of HSC and MPP1 8-wk-old mice were treated with a single injection of 5-FU, and the cell cycle status 5 d after 5-FU treatment. Indeed, more Dok DKO BM was analyzed after 3 or 5 d for cell cycle status and cellularity. than WT HSC exit quiescence (G0) and enter cell cycle (S-G2-M) We found that BM cellularity in Dok DKO mice was increased as during HSC/MPP1 transition (Fig. 6E). This results in a reduced compared with WT mice with a dramatic increase of LK and LSK proportion of DKO HSC in G1, suggesting that these cells had fractions after 3 d (Fig. 6A). Five days after 5-FU injection, the already proceeded to MPP1 differentiation. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Dok1 and Dok2 proteins control hematopoietic progenitors quiescence. (A) DNA content (DAPI) versus Ki67 staining of LSK (Lin2,c-Kit+, Sca-1+) and MPP3/4 cells (CD1502,CD48+LSK) in WT and Dok DKO mice shows cell cycle phase represented by each quadrant. Histograms show percentage mean of LSK an LK cells. (B and C) LSK cells compartments from HSC (CD342,CD150+,CD482 LSK), MPP1 (CD34+,CD150+,CD482 LSK), + + 2 + MPP2 (CD150 ,CD48 LSK), and MPP3/4 (CD150 ,CD48 LSK) in G0,G1, and S-G2-M phases of cell cycle. Data are from the FACS plots above. Mice are 6–8 wk old. Representative results of at least three independent experiments with n =6.*p , 0.05, **p , 0.01. (D) Heat-map representation of gene expression in the indicated subset isolated from Dok DKO or WT control mice. Results obtained with bulks of 10 sorted cells are shown. Color code indicates relative expression with respect to Actb signals normalized to 1.

Dok1 and Dok2 deficiency protects against 5-FU of Dok DKO mice to 5-FU–induced exhaustion. This would be in myeloablative treatment agreement with increased proportion of erythroid progenitors Increased hematopoiesis of Dok DKO mice after myeloablation observed in Dok DKO mice at steady state (Supplemental Fig. 1C, should result in an enhanced sensitivity to sustained aplasia, but 1D). Altogether, our results reveal an unexpected function for Dok the rapid re-entry of mutant HSC in a quiescent state compared proteins in the control of cell cycle regulation at the transition with WT cells may have a protective role on the HSC pool. To checkpoint between HSC and MPP1 resulting in an overall higher address this issue, mice were subjected to weekly 5-FU injections hematopoietic regenerative capacity of Dok DKO mice in re- and their survival was monitored over time. WT mice survival was sponse to myeloablative stress. significantly reduced compared with Dok DKO littermates (Fig. 7A). More than 80% of WT mice died compared with only Discussion 6% of Dok DKO mice. Peripheral blood monitoring during the Previous reports have shown that Dok1 and Dok2 are involved in experiment showed that there were more WBC (lymphocytes, myeloid homeostasis and leukemia suppression due to their neg- monocytes, and reticulocytes) in Dok DKO mice as compared ative impact on signaling pathways involved in hematopoietic with WT mice (Fig. 7B). This was associated to a marked re- growth and differentiation (8, 11). To investigate the role of these duction of anemia, which may account for the increased resistance proteins, the double Dok1- and Dok2-deficient mice are a useful 8 Dok1 AND Dok2 REGULATE HSPC CELL CYCLE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Dok1 and Dok2 proteins control cell cycle and numbers of circulating myeloid cells after single 5-FU injection. (A)Kineticsofperipheralblood leukocyte counts after 120 mg/kg/i.p. 5-FU first (day 0) and second (day 21) injections. (B) Histogram representatives of peripheral blood populations absolute numbers at day 0 (left panel) and day 14 (right panel) after 5-FU first injection. (C) Top panel, FACS gating strategy of peripheral blood monocytes (CD11b+,Gr-1lo); left panel, Ki67 staining of gated monocytes. Monocytes are from WT and Dok DKO mice, 9 d after 5-FU injection. Bottom panel, Representative histograms of percentage of cells positive for Ki67 marker in each peripheral blood stained population after 3, 6, and 9 d post–5-FU injection. Counts were performed by FACS with counting beads (A and B). Mice were 8 wk old. Results are representative of two independent experiments with n = 7 or 8. Mann–Whitney t test, *p , 0.05, **p , 0.01, ***p , 0.001. animal model because they are overlapping functions of DOK1 Loss of Dok1 and Dok2 leads to increased BM cell proliferation with its closest family member, DOK2 (34). DOK1 and DOK2 are (6, 7). Although this suggests a role for the Dok proteins in the docking proteins and are highly related in structure (34, 37). maintenance of hematopoietic homeostasis, this issue has never The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. After a single 5-FU injection, Dok1 and Dok2 proteins regulate BM cellularity, cell cycle entry, and MPP1 numbers. (A) Dok DKO and WT mice were injected with 5-FU (120 mg/kg/i.p.). Whole BM count (left panel); Lin2 gated cells (middle panel) and histogram representative of LK and LSK populations per- centage, 3 d after 5-FU injection (120 mg/kg/i.p.); n =3.(B) Five days after 5-FU injection, counts (left panel) and CFU-GM (middle panel) of total BM were assessed. Absolute numbers of LSK subsets (right panel); results are representative of two independent experiments with n =6.Mann–Whitneyt test, *p , 0.05, **p , 0.01,

***p , 0.001. Percentage of HSC and MPP1 cells in G0 cell cycle stage, 3 d (C)and5d(D) after injection. n = 9; Mann–Whitney t test, *p , 0.05, ***p , 0.001. (E) Percentage variation of cells in G0 (left panel), G1 (middle panel), and S-G2-M (right panel) cell cycle stages from HSC to MPP1 progenitors in WT compared with Dok DKO mice 5 d after 5-FU injection. n = 9. Results are representative of two independent experiments. Paired t test; *p , 0.05, **p , 0.01, ***p , 0.001. been addressed before. In this study, we demonstrate that Dok1 crease of quiescent G0-phase HSC is detected in WT mice be- and Dok2 proteins regulate cell cycle at the transition between tween days 3 and 5 (Fig. 6). This drop in the number of quiescent HSC and MPP1. After a single 5-FU challenge, a dramatic de- HSC is not detected in Dok DKO mice, indicating that Dok1 and 10 Dok1 AND Dok2 REGULATE HSPC CELL CYCLE Downloaded from

FIGURE 7. Dok1 and Dok2 induce resistance to 5-FU induced HSC exhaustion. (A) Dok DKO and WT mice (n = 17) were injected weekly with 5-FU (120 mg/kg/i.p.), and survival was moni- tored. Gehan-Breslow-Wilcoxon test; p , 0.0001.

(B) Monitoring of peripheral blood leukocytes and http://www.jimmunol.org/ RBCs parameters in 5-FU weekly–injected mice. n = 7 at the beginning of experimental protocol. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 26, 2021

Dok2 are negative regulators of proliferative signals during BM AKT signals (34). They have been reported to block RAS/ERK regeneration and may act as brakes counterbalancing microenvi- and PI3K/AKT pathways in murine myeloid cells (6, 7, 40). In ronmental cues maintaining HSC quiescence. addition to these posttranslational modifications, here we show At steady state, Dok1 and Dok2 control progenitor cellularity by that loss of Dok1/Dok2 expression results in upregulation of negative regulation of cell cycle. Indeed, Dok DKO mice showed Cdkn1a (p21cip1) gene expression (Fig. 4), reported to be in- increased proportions of cells in G1 and/or S-G2-M phases of cell volved both in negative or positive role in HSPC pool size (26, cycle in LSK and LK cells (Fig. 4). In human erythroid progen- 41). Moreover, Dok1/Dok2 deficiency induces a downregula- itors, Dok1 phosphorylation has been correlated with a down- tion of Cdkn1c (p57kip2)geneexpressioninHSC,knownasa regulation of CDK inhibitors (8), CDKN1A (p21cip1)and master regulator of HSC quiescence (27, 42). Furthermore, CDKN1B (p27kip1), suggesting that Dok1 is involved in negative Dok1/Dok2 double deletion is also associated to Mpl gene regulation of molecules involved in cell cycle arrest. Common downregulation. TPO/MPL signaling is required for the regu- signaling pathways such as RAS/ERK pathways are involved in lation of HSC quiescence and is associated with upregulation of cell cycle regulation (38, 39). DOK1 and DOK2 proteins block genes encoding for CDK inhibitors in HSC (43). Altogether, these signals by recruiting enzymes, RasGAP and SHIP-1, re- these data suggest a broader effect of Dok1 and Dok2 in HSC spectively, involved in negative regulation of RAS/ERK and PI3K/ cell cycle regulation. The Journal of Immunology 11

Serial transplantation experiments show a progressive decrease progenitors. Paradoxically, upon hematopoietic stress, Dok pro- of WT HSC compared with Dok DKO HSC (Fig. 2), suggesting teins oppose the quiescence of HSC (8, 9, 48, 49). Dok1 and Dok2 that serial transplantations generate a lower rate of HSC exhaus- dysregulations have been associated with several oncogenic tion in Dok DKO context. Dok1/Dok2 deficiency is associated processes (1, 6, 7, 12, 50–56). Therefore, the impact of chemo- with Gata3 gene downregulation at HSC stage (Fig. 4D). Similar therapeutic agents on HSCs may have to be considered to avoid to Dok DKO HSC (Fig. 2F), an expansion of Gata3-deficient resistance of leukemic stem cells and subsequent relapse. HSC is reported after serial transplantations showing that Gata3 is associated with increased HSC self-renewal (44). These re- Acknowledgments sults are supported by the differential sensitivity of Dok DKO We thank V. Ferrier-Depraetere (Institut Paoli-Calmettes) for thoughtful mice as compared with control after serial 5-FU injections reading of the manuscript. We thank M.-L. Thibult and F. Mallet for assis- (Fig. 7). Indeed, 5-FU myeloablation induces more all LSK sub- tance with the use of the cytometry and cell sorting facility; P. Gibier, sets (including HSC) in G1 and S-G2-M phases of cell cycle J.-C. Orsoni, and F. Gianardi for animal facilities; E. Agavnian for work (Supplemental Fig. 3B). Thus, in agreement with the finding that on the Institut Paoli-Calmettes/Centre de Recherche en Cance´rologie de genes involved in cell cycle regulation are mostly affected in HSC Marseille experimental pathology platform; C. Fauriat, C. Acquaviva, compartment of Dok DKO mice, it seems that Dok1 and Dok2 S. Sarrazin, F. Brunet, D. Birnbaum, E. Duprez, and P. Dubreuil for helpful negatively regulate cell cycle at the transition between HSC and discussions; and F. Bardin, S. Morin, and A. Goubard for helping to con- MPPs. Moreover, the cell cycle status of HSC and MPP1 pro- duct the mouse experiments. genitors in G0 and S-G2-M state suggests that Dok DKO HSC proceeds faster to MPP1 differentiation (Fig. 6E), suggesting that Disclosures Downloaded from cell proliferation and differentiation may represent two sides of The authors have no financial conflicts of interest. the same coin. The percentage of G1-phase HSC and MPP1 cells is higher in Dok DKO than in WT mice. Because transition from References HSC to MPP1 is increased in Dok DKO mice compared with 1. Carpino, N., D. Wisniewski, A. Strife, D. Marshak, R. Kobayashi, B. Stillman, WT cells, these results suggest that a process is engaged to bring and B. Clarkson. 1997. p62(dok): a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells. back Dok DKO HSC to a quiescent state. 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60

40 C-kit MEP % of CMP of % CD16/32 20 CMP 0

Sca-1 CD34 0- D105- CD105-15 C CD CD150+

GMP CMP MEP 5

4 29%

69% ) 3 5

(.10 2 CD105 1 Absolute cell number cell Absolute 0 CD150 5- 05- D10 50+ D1 1 C D150- C C CD C Total BM D WT DKO

50 WT 1.5 2%II 40% 1% 39% DKO ** I I II 40 ) 9 1 30 1.4% 3.3% (.10 III 20 CD71 III 0.5

% of BM cells BM of % * ** 9.2% 12% ** ** 10 number cell Absolute IV IV 0 0 I I I I I I II IV I III IV Ter119 Supplementary Figure 1: Efects of Dok1 and Dok2 double defciency on CMP and erythroid progenitor subsets (A) Upper panel: Gating strategy of CMP (CD16/32-, CD34+), GMP (CD16/32+, CD34+) and MEP (CD16/32-, CD34-) gated on lineage- C-kit+ Sca-1- cells. Lower panel: CD105 and CD150 diferential expression of GMP, CMP and MEP. CMP can be subdivided in 2 subpopulations: CD105- CD150- and CD105- CD150+ cells. (B) Percentages (upper panel) and absolute numbers of CMP cells (lower panel) with the indicated phenotype. (C) Representative dot-plots for CD71/Ter119 staining on total bone marrow of wild type and Dok DKO mice. CD71 and Ter119 expression allows identifcation of four major erythroid progenitor subsets as indicated: region I, pro-erythroblasts (CD71Hi Ter119Lo); region II, basophilic erythroblasts (CD71Hi Ter119Hi); region III, chromatopholic erythroblasts (CD71Lo Ter119Hi); and region IV, orthochromatophilic erythroblasts (CD71- Ter119Hi). (D) Quantifcation of percentages and absolute numbers of erythroid progenitors according to the above classifcation. Mice are 6-8 weeks old. Representative results of at least 3 independent experiments with n = 6 mice/group. * P<0.05; ** P<0.01. n=6. * P<0.05; ** P<0.01. 6 9,5Gy A 5.10 6 9,5Gy 5.10 6 9,5Gy 5.10 total BM total BM total BM

12 weeks 12 weeks 12 weeks DKO or WT F1(C57Bl/6x129/sv) 129/Sv CD45.2 = CD45.1/2 n = 10

Mature (BM, Blood, Spleen) and progenitor cells phenotype

B Blood Spleen BM Blood Spleen BM 100 100 100 2% 4% 1.1% 98 98 98 Recipient

96 96 96

94 94 94 CD45.1

Donor % of CD45.2 cells CD45.2 of % 92 92 92 98% 94% 94% 90 90 90 T O T O WT DKO W DK W DK CD45.2

C D BM Spleen Blood Spleen BM

WT 6 8 60 60 80 ** ** DKO 6 60 4 40

) 40 1st 7 10

. 4 40 (

% of cells of % 2 20 20 *** 2 * 20 Cell absolute number absolute Cell

0 0 0 0 0 i i + lo + lo + + lo WT WT 1 9+ b h 3 9 1 DKO DKO 19 1 r1 1 1b+ r1h r CD3+ D CD3+ G CD D G C D11b+ CD - Gr1 C D1 C CD11 0 C 8 80- G 0+ / /80+ 8 4 4 4/ F4/80- Gr1hiF4/80+ Gr F F F4/ F

10 *** 10 60 60 80 *** ** *** *** 8 8 ** 60 40 40 * 6 6 ) 7 40 ** 2nd 10 4 4 . (

20 20 % of cells of % ** 2 *** *** 20 2 ** *

Cell absolute number 0 0 0 0 0 + + + i + i 3 9 b lo lo 3 h lo WT WT 1 r1 r1 1b+ r1 1 DKO DKO CD D1 G CD3+ D19+ G CD 1 G C - Gr1h C CD19+ - Gr CD1 0 + CD11b+ + CD 0 + 8 0 0 8 0 /8 / /8 4 4/8 4 4 F4/ F F4/80- Gr1hiF F F Supplementary Figure 2: Features of Dok DKO mice are transplantable and cellular autonomous. (A) Schematic representation of the protocol used for serial transplantations. (B) Left: Percentage of chimerism observed in peripheral blood, spleen and bone marrow of lethally irradiated mice, 12 weeks after engraftment with WT or Dok DKO bone marrow. Dok1-/- Dok2-/- cells are CD42.2 whereas the recipient cells are double positives for CD45.1 and CD45.2 markers. Right: Representative fow cytometry profles showing chimerism of the previous hematopoietic organs (n=10 for each genotype). (C) Cellularity of bone marrow and spleen of frst and secondary transplanted mice with Dok DKO or WT bone marrow cells. (D) Monocytosis and increase of progenitors numbers of Dok1-/- Dok2-/- DKO mice is transplantable and cell autonomous. Results obtained in peripheral blood, spleen and bone marrow from primary (top panels) and secondary recipient mice (lower panels) are shown. n=10. * P<0.05; ** P<0.01; *** P<0.001. Dok DKO: black bar, wild type: white bar. A WT DKO

NT

100 µm 100 µm

5 days post 5-FU

100 µm 100 µm

B WT LSK WT HSCs

1.1% 51.4% 16.2% 43.4%

29.5% 45.5%

Ki67 DKO LSK Ki67 DKO HSCs

21.8% 4.5% 55.5% 50.5%

21.9% 28.3%

DAPI DAPI

Supplementary Figure 3 Changes in bone marrow cellularity and HSPC cell cyle after 5-FU Injection in WT and Dok DKO mice. (A) Hematoxylin Eosin Safran (HES) staining of bone marrow sections obtained from WT and Dok DKO BM at steady state (NT: Non treated on the top) and 5 days after 5-FU induced myeloablation (bottom). (B) Representative fow cytometry profles of Ki67 versus DAPI staining obtained on total LSK (Left panels) and HSC subsets (right panels) 3 days after 5-FU injection (120 mg/kg/ip). Supplementary Table: Taqman probes used in this study

Actb Mm00607939_s1 Gapdh Mm99999915_g1

Hprt Mm01545399_m1 Dok1 Mm01184860_g1 Dok2 Mm01315632_m1 Csf2ra Mm00438331_g1 Cdkn2a Mm00494449_m1 Ccne2 Mm00438077_m1 Mpl Mm00440310_m1 Rbl1 Mm01250721_m1 Kit Mm00445212_m1 Myb Mm00501741_m1 Cdkn1a Mm00432448_m1 Cdkn1c Mm00438170_m1 Cdk6 Mm01311342_m1 Cdkn2d Mm00486943_m1 Ccnf Mm00432385_m1 Ccnb2 Mm01171453_m1 Rb1 Mm00485586_m1 Gata3 Mm00484683_m1 Ccne1 Mm01266311_m1 Cdkn1b Mm00438168_m1 Ccnd1 Mm00432359_m1 Ccnd2 Mm00438070_m1