Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.

Article

Estrogen Signaling Selectively Induces Apoptosis of Hematopoietic Progenitors and Myeloid Neoplasms without Harming Steady-State Hematopoiesis Graphical Abstract

Authors Abel Sańchez-Aguilera, Lorena Arranz, ..., Ju¨rg Schwaller, Simoń Meńdez-Ferrer

Correspondence [email protected] (A.S.-A.), [email protected] (S.M.-F.)

In Brief In mice, activation of estrogen receptor (ER) signaling induces apoptosis in multipotent hematopoietic progenitors but induces proliferation and compromises function of HSCs in a reversible manner. Tamoxifen induces apoptosis of MLLAF9+ blasts, improving conventional chemotherapy, and tamoxifen alone blocks JAK2V617F-induced myeloproliferative neoplasm by restoring normal apoptosis levels in mutant cells.

Highlights

Accession Numbers

Hematopoietic stem and multipotent progenitor cells (MPPs) express ERa

GSE62621

ERa activation induces apoptosis of MPPs and proliferation of LT-HSCs Tamoxifen blocks neoplasia in mice

JAK2V617F-induced

myeloproliferative

Tamoxifen enhances chemotherapy response of MLL-AF9induced leukemias

Sańchez-Aguilera et al., 2014, Cell Stem Cell 15, 791–804 December 4, 2014 ª2014 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2014.11.002

Cell Stem Cell

Article Estrogen Signaling Selectively Induces Apoptosis of Hematopoietic Progenitors and Myeloid Neoplasms without Harming Steady-State Hematopoiesis Abel Sańchez-Aguilera,1,* Lorena Arranz,1 Daniel Martıń-Pe´rez,1 Andre´s Garcıá-Garcıá,1 Vaia Stavropoulou,2 Lucia Kubovcakova,2 Joan Isern,1 Sandra Martıń-Salamanca,1 Xavier Langa,1 Radek C. Skoda,2 Ju¨rg Schwaller,2 and Simoń Meńdez-Ferrer1,* 1Stem

Cell Niche Pathophysiology Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid 28029, Spain of Biomedicine, University Hospital Basel, CH-4031 Basel, Switzerland *Correspondence: [email protected] (A.S.-A.), [email protected] (S.M.-F.) http://dx.doi.org/10.1016/j.stem.2014.11.002 2Department

SUMMARY

Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERa activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2V617F-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2V617F+ HSPCs in a xenograft model, and sensitized MLL-AF9+ leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs. INTRODUCTION In adult mammals, hematopoietic stem cells (HSCs) reside in a complex bone marrow (BM) microenvironment where they respond to multiple extracellular signals (Mercier et al., 2012). HSCs are associated with perivascular niches (Kiel et al., 2005; Meńdez-Ferrer et al., 2010) and are exposed to signals transported by the circulatory system, including hormones. Estrogens are potential HSC niche modulators given their bone anabolic properties (Colaianni et al., 2012; Compston, 2001; Hughes et al., 1996; Nakamura et al., 2007). Estrogens regulate lymphopoiesis. ERa is expressed by B cell precursors (Smithson et al., 1995), thymocytes (Kawashima

et al., 1992), and CD8+ T cells (Stimson, 1988), while ERb is expressed in thymus and spleen (Kuiper et al., 1997; Mosselman et al., 1996). Mice deficient in ERa have fewer BM B cells, whereas pharmacological treatment with the natural ER agonist 17b-estradiol (E2) depletes lymphoid-restricted progenitors and B/T cell precursors (Medina et al., 2001; Thurmond et al., 2000). Data regarding effects of estrogens on more primitive hematopoietic cells are somewhat conflicting. E2 was initially reported to decrease lin c-kit+ Sca-1+ (LSK) HSPCs in mouse BM (Thurmond et al., 2000) but increase the number of vascular-associated HSPCs, likely through the regulation of stromal cells (Illing et al., 2012). HSC division frequency is higher in female mice than in male mice, and it is further increased by exogenous E2 administration or pregnancy, although these effects did not change BM HSC numbers. In addition, estrogen signaling promotes extramedullary hematopoiesis and erythropoiesis in the spleen (Nakada et al., 2014). Aberrant estrogen signaling might contribute to leukemogenesis because mice deficient in ERb develop chronic myeloproliferative disease during aging (Shim et al., 2003). Moreover, women consistently exhibit lower incidence of hematological cancers compared to men (American Cancer Society, 2013; Cancer Research UK, 2013), strongly suggesting a contribution of sex hormones in the development of such malignancies. However, whether estrogen signaling can directly control normal and leukemic HSPCs has remained unclear. Outside the hematopoietic system, ER activation stimulates the growth of solid tumors, most prominently in breast cancer (Adkins et al., 1999; Rodenhuis et al., 2003; Tallman et al., 2003; Wang et al., 2004b), and the selective ER modulator (SERM) tamoxifen is clinically used to treat ER+ breast cancer and recurrent gliomas. In this study we found that estrogen signaling differentially regulates survival, proliferation, and self-renewal of normal and malignant HSPCs. Furthermore, our data demonstrate that therapeutic targeting of this pathway with the SERM tamoxifen could be a valuable strategy for blocking the development of chronic myeloproliferative neoplasms (MPNs). RESULTS Expression of ERs in Hematopoietic Progenitors To determine whether HSPCs might be directly regulated by estrogens, ER expression was analyzed in purified hematopoietic

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Cell Stem Cell Regulation of Hematopoiesis and MPN by Estrogens

cells. Esr1 (ERa) and Esr2 (ERb) mRNA and proteins were differentially expressed by distinct HSPC populations. Intriguingly, Esr1 expression was very high in long-term HSCs (LT-HSCs) and multipotent progenitors (MPPs) and lower in committed progenitors and mature lineages. In contrast, Esr2 expression was very low or undetectable (Figures 1A–1C), in agreement with public gene expression data (Figure S1A available online). Esr1 expression was similar in HSPCs from male and female mice (data not shown). ERa-deficient (Esr1 / ) mice had reduced BM LT-HSCs and short-term HSCs (ST-HSCs), but similar levels of MPPs, megakaryocyte precursors (lin c-kit+ Sca-1 CD41+), and CD41+ megakaryocytic cells (Figure 1D and data not shown). However, ERa or ERb were not required for long-term multilineage reconstitution after BM transplantation, and Esr1 / BM cells exhibited only a partial reduction in short-term engraftment (Figures 1E and 1F). This suggested differential roles of estrogen signaling in specific HSPC subsets. Estrogen Signaling Differentially Regulates Survival and Proliferation of Hematopoietic Progenitors We administered tamoxifen to male mice to avoid the confounding effects of variably high endogenous estrogens in females. Surprisingly, treatment of WT mice with tamoxifen at a dose typically used to induce CreER recombinases caused a rapid reduction in BM cellularity (50%–60% of control as early as 1 week; Figure 1G). However, except for reduced platelet counts, most hematological parameters were normal in mice chronically treated with tamoxifen (Table S1 available online), suggesting mild effects on mature hematopoietic cells. Tamoxifen rapidly reduced levels of LSK cells (Figures 1H and S1B), preceding the decay in BM cell number. Interestingly, differential effects were observed in distinct HSPC populations. While MPPs were 7-fold reduced, the decrease in ST-HSCs and more mature myeloid progenitors was less pronounced and LT-HSC numbers were relatively preserved (Figures 1I–1J and S1B). Further subfractionation revealed that MPP4 and MPP3 (Wilson et al., 2008) were the most affected populations. In contrast, the number of LT-HSCs, defined as LSK CD150+ CD48 or LSK CD150+ CD48 CD34 CD135 cells, did not change after treatment (Figure S1C and data not shown). These effects were the result of specific changes in cell survival and proliferation. ST-HSC/MPP depletion by tamoxifen was caused by apoptosis, whereas LT-HSCs and myeloid progenitors were more resistant to apoptosis (Figure 1K; see also Figure 5D). In a manner similar to that in males, tamoxifen also decreased BM HSPCs by apoptosis in female mice (Figures S1D and S1E). A more pronounced apoptotic effect on LTHSCs was observed in females, which might be related to their higher proliferation rate (Nakada et al., 2014). Treatment of BM lin cells in vitro with 4-hydroxytamoxifen (4OHT) also induced apoptosis, suggesting a direct proapoptotic effect of tamoxifen on HSPCs (Figure S1F). While LT-HSCs were comparatively more resistant to tamoxifen-induced cell death, their proliferative fraction increased after 2 weeks of treatment (Figure 1L). These results indicate that survival and proliferation are differentially regulated by estrogens in distinct HSPC subsets.

The Effects of Tamoxifen on HSPCs Are Direct and Mediated by ERa Gene expression profiling was performed by next-generation sequencing (RNA-seq) in LT-HSCs, ST-HSCs, and MPPs isolated from tamoxifen- or vehicle-treated mice. Principal component analysis showed a coherent clustering of biological replicates (Figure 2A), indicating that tamoxifen treatment did not cause a global deregulation of gene expression profiles. Instead, tamoxifen caused coordinated changes in the expression of genes associated with HSC self-renewal, cell cycle, signal transduction, and metabolism, as revealed by gene-set enrichment analysis (GSEA) (Krivtsov et al., 2006; Subramanian et al., 2005). Few changes were common to all three HSPC populations, consistent with their differential in vivo responses. Most differentially expressed gene sets were downregulated in LT-HSCs but upregulated in ST-HSCs and/or MPPs (Figures 2B and S2). Tamoxifen can function as an ER agonist/antagonist in a cell-type- and context-dependent manner. To characterize its ligand activity in HSPCs, we analyzed how tamoxifen affected the expression of genes positively or negatively regulated by the native ER agonist E2 in other tissues (Frasor et al., 2003; Ghosh et al., 2000; Laganie`re et al., 2005; Wang et al., 2004a). Globally, these genes exhibited similar changes in LT-HSCs upon tamoxifen treatment—E2-induced genes were upregulated and E2-repressed genes showed decreased expression—suggesting an ER agonist action of this SERM on LT-HSCs (Figure 2C). E2-induced genes were upregulated by tamoxifen in all three HSPC populations, but E2-repressed genes were specifically downregulated in LT-HSCs (Figure S2), which could be related to reduced levels of ER corepressors in ST-HSCs/MPPs compared to LT-HSCs (Figure S3A). These results suggest that tamoxifen has ER agonistic activity in HSPCs, where it activates or represses genes in a cell-typespecific manner. To confirm that these effects were mediated by canonical ER signaling, pharmacological studies were performed in mice lacking ERs. Tamoxifen reduced ST-HSC and MPP numbers by apoptosis in control and Esr2 / , but not in Esr1 / , mice. In addition, LT-HSC cycle entry caused by tamoxifen was mostly abolished in the absence of ERa (Figures 2D–2F). In order to clarify whether these effects were direct or mediated by the BM microenvironment, WT recipient mice were transplanted with WT or Esr1 / BM cells, allowed to engraft for 4 months, and treated with tamoxifen or vehicle for 2 weeks. Tamoxifeninduced changes were not observed in mice with Esr1 / hematopoiesis and thus were hematopoietic cell autonomous (Figures 2G and 2H). Given the reduction in platelet counts observed in treated mice, we analyzed the effect of tamoxifen on megakaryocytic precursors. This population was also depleted by apoptosis in an ERa-dependent, cell-autonomous fashion (Figures 2G and 2H). These data indicate that tamoxifen can directly regulate hematopoietic cell proliferation and survival through the activation of ERa expressed by HSPCs. ERa Activation Alters Gene Expression Patterns Related to HSC Self-Renewal, Proliferation, and Apoptosis LT-HSCs display a low proliferative rate (Morrison et al., 1995), which allows for their long-term self-renewal (Wilson et al.,

792 Cell Stem Cell 15, 791–804, December 4, 2014 ª2014 Elsevier Inc.


Figure 1. Differential Regulation of HSPC Survival and Proliferation by Estrogen Signaling (A and B) Relative mRNA expression of Esr1 and Esr2, measured by qPCR and normalized to that of Gapdh, in murine hematopoietic cell subsets. (C) Representative western blot of Esr1 and Esr2 in sorted HSPCs. Mouse ovary, positive control. Values indicate densitometric ratios of Esr1/Actb, normalized to the ovary sample. (D) HSPC numbers in the BM of male WT, Esr1 / , or Esr2 / mice. (E and F) Competitive repopulation capacity of ER-deficient BM cells, measured over time (E) as donor (CD45.2+) chimerism in peripheral blood and (F) as multilineage reconstitution in BM 11 months posttransplant. Lethally irradiated recipients were transplanted with 106 WT or ER null BM cells and 106 B6.SJL (CD45.1+) competitor BM cells. (G) Number of total BM nucleated cells, (H) LSK cells, (I) HSPC subpopulations, (J) colony-forming units in culture (CFU), (K) fraction of apoptotic (Annexin V+) cells in HSPC subpopulations, and (L) cell-cycle distribution of LTHSCs in the BM of male WT C57BL/6J mice after in vivo tamoxifen treatment for the indicated time frame (J, 8 days). Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Cell numbers correspond to two hind legs (G) or four limbs, sternum, and spine (D, H, and I). n = 3–6 (D–F), n = 5 (G, J, and K), n = 2–7 (H, I, and L). Data in (A)–(F) show one of two independent experiments; (G)–(L) are representative of three or more experiments. CMP, common myeloid progenitors; CLP, common lymphoid progenitors; CD19, B cells; CD90, T cells; Gr1-Mac1, mature myeloid cells; Blast, MLL-AF9+ AML blasts; FL, fetal liver; TAM, tamoxifen. See also Figure S1 and Table S1.



Figure 2. The Effects of Tamoxifen on HSPCs Are Mediated by ERa Expressed by Hematopoietic Cells (A) Principal component analysis of gene expression profiling data (RNA-seq) of LT-HSCs, ST-HSCs, and MPPs from tamoxifen-treated or control male C57BL/6J mice. Each dot represents one sample pooled from two mice. n = 3 replicates per condition. (legend continued on next page)



2008). In parallel with their increased proliferation, a striking feature of LT-HSCs from tamoxifen-treated mice was the loss of gene expression patterns associated with self-renewal and HSC identity (Figures 3A and S2 and Table S2 and Table S3). GSEA revealed coordinated downregulation of the self-renewal-associated signature described for HSCs and leukemia stem cells, the HSC-specific ‘‘fingerprint,’’ and genes positively regulated by the polycomb ring-finger oncogene Bmi1; conversely, LT-HSCs from tamoxifen-treated mice showed increased expression of genes repressed in self-renewing cells and negatively regulated by Bmi1 (Arranz et al., 2012; Chambers et al., 2007; Krivtsov et al., 2006). Tamoxifen treatment also activated the transcriptional program of the ER target Myc (Zeller et al., 2006), associated with HSC differentiation and loss of self-renewal (Wilson et al., 2004). Indeed, Myc and Myc-induced genes were upregulated in tamoxifen-treated HSCs, whereas the expression of Mycrepressed genes was mostly reduced (Figures 3B and 3C), coincident with the induction of cell cycle progression genes (Figure 3D). In addition, many of the genes upregulated by tamoxifen in HSPCs were Myc targets (Figure S3B). Expression of the chromatin remodeling factor Satb1, which represses Myc and is required for HSC quiescence and self-renewal (Will et al., 2013), was significantly downregulated by tamoxifen in both LTand ST-HSCs (Figure 3E). To determine whether HSPC changes elicited by tamoxifen required Myc function, conditional Mycdeficient mice (Pol2-creERT2;MycloxP/loxP) were treated with tamoxifen chronically or only transiently, to activate CreERT2. Myc-deficient MPPs did not undergo cell death upon chronic tamoxifen treatment (Figure 3F), suggesting that Myc mediates, at least partially, tamoxifen’s effects on HSPCs. In addition, tamoxifen reduced the expression of the stem cell factor receptor Kit specifically in LT-HSCs; c-kit protein levels were also reduced by tamoxifen in the cell surface of WT or Esr2-deficient LT-HSCs, but not in Esr1-deficient LT-HSCs (Figures 3G and 3H), indicating that this effect was also mediated by ERa. Consistent with our initial findings, ST-HSCs (but not LTHSCs) from tamoxifen-treated mice showed increased expression of proapoptotic genes (Aifm1 and Casp3) and reduced levels of antiapoptotic genes (Cflar) (Figure S4A). These changes were not observed in MPPs, probably due to their earlier apoptosis and depletion (Figure S1B) or the selection of estrogen-resistant subpopulations (MPP1 and MPP2; Figure S1C). Thus, tamoxifen impairs the self-renewal program of LT-HSCs and induces apoptosis of more differentiated progenitors, at least partially via ERa-mediated induction of Myc.

Tamoxifen Compromises Hematopoietic Recovery after Myelotoxic Stress To determine whether tamoxifen-induced changes in LT-HSC proliferation and self-renewal might have functional consequences, we studied the hematopoietic recovery of mice treated with tamoxifen or vehicle and subsequently subjected to myelotoxic regimens. Tamoxifen-treated mice exhibited defective multilineage hematopoietic recovery after 5-fluorouracil (5-FU) challenge (Figure 3I) and premature death upon sublethal irradiation (Figure S4B). To study the reversibility of these effects, LT-HSCs from tamoxifen-treated mice were transplanted into tamoxifen-free recipients. These cells exhibited unaltered capacity for long-term multilineage reconstitution in a normal environment, demonstrating that the effects of tamoxifen can revert in the absence of the ligand (Figure S4C). In order to investigate whether the detrimental effect of tamoxifen on HSC function was also dependent on ERa, we studied the hematopoietic recovery of tamoxifen/vehicle-treated Esr1 / mice after 5-FU administration. Unlike WT mice, tamoxifentreated Esr1 / mice exhibited normal reconstitution kinetics. Thus, tamoxifen’s effects on HSC function are also mediated by ERa (Figure 3I). Tamoxifen Blocks the Development of JAK2V617F-Induced MPNs The differential effects of ER activation in HSPCs suggested a potential use of SERMs to treat hematological malignancies through a dual action: impairment of leukemia-initiating cells and reduction of leukemic burden through apoptosis of leukemic progenitors. Therefore we tested the potential therapeutic effect of tamoxifen in a mouse model expressing a point mutation of the Janus kinase 2 (JAK2V617F) in HSCs, frequently found in patients with MPNs (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; Levine et al., 2005). Male mice transplanted with polyI-polyC-induced Mx1-cre;JAK2V617F BM cells, which develop polycythemia vera-like MPN (Kubovcakova et al., 2013; Tiedt et al., 2008), and disease-free control mice were chronically treated with tamoxifen when granulocytosis and erythrocytosis were detected in peripheral blood (Figure S5A). Notably, tamoxifen treatment completely abolished MPN-associated neutrophilia and thrombocytosis, with no major side effects in control mice (Figure 4A). Tamoxifen treatment also alleviated the early increase in red cell counts, hemoglobin, and hematocrit; decreased BM megakaryocytes; and reduced MPN-associated splenomegaly, with preservation of spleen architecture and decreased infiltration of interfollicular areas by megakaryocytes (Figures 4A–4C and S5B–S5F). Prolonged

(B) Gene sets significantly changed (FDR < 0.15) in at least one HSPC population after tamoxifen treatment. Red, blue, and gray indicate upregulation, downregulation, and no change, respectively, in tamoxifen-treated HSPCs. Figure S2 shows an extended version of this diagram. (C) Gene set enrichment analysis of estrogen target genes (genes previously reported to be induced or repressed by E2) in LT-HSCs from C57BL/6J mice treated with tamoxifen/vehicle for 2 weeks. (D–F) Number of (D) ST-HSCs/MPPs (per four limbs, sternum, and spine), (E) cell-cycle distribution of LT-HSCs, and (F) fraction of apoptotic ST-HSCs/MPPs in the BM of male WT, Esr1 / , or Esr2 / mice after in vivo tamoxifen/vehicle treatment for 2 weeks. (G and H) Number of (H) total BM nucleated cells and HSPC subsets, and (G) fraction of apoptotic HSPCs in the BM of male WT C57BL/6J mice transplanted with WT or Esr1 / BM cells, engrafted for 4 months and treated with vehicle/tamoxifen for 2 weeks. Data are representative of two independent experiments. Data are means ± SD. n = 2–7 (D–F), n = 5 (G and H). *p < 0.05, **p < 0.01, ***p < 0.001 (vehicle versus tamoxifen). ES, enrichment score; NES, normalized enrichment score; FDR, Benjamini-Hochberg false discovery rate; MkP, megakaryocyte precursors (lin c-kit+ Sca-1 CD41+). See also Figure S2, Table S2, and Table S3.



Figure 3. Tamoxifen Compromises HSC Self-Renewal and Activation during Stress Hematopoiesis (A, B, and D) Coordinated changes, determined by gene-set enrichment analysis, in genes associated with (A) HSC self-renewal, (B) Myc-regulated transcriptome, and (D) cell-cycle progression in LT-HSCs isolated from C57BL/6J mice treated with tamoxifen/vehicle for 2 weeks. (C and E) Expression levels of Myc and Satb1 genes in BM HSPCs from C57BL/6J mice treated as in (A). Data are fragments per kilobase of exon per million mapped reads (FPKM) ± SD. n = 3. Adjusted *p < 0.15, **p < 0.05. (F) Number of MPPs (per hind leg) in the BM of male Pol2-creERT2;MycloxP/loxP or control mice treated with tamoxifen for 4 weeks. Horizontal lines indicate the mean. n = 4. (G) mRNA expression levels of Kit, determined as in (C) and (E). (H) Surface c-kit protein level measured by flow cytometry in immunophenotypically defined LT-HSCs from WT or ER-deficient mice. Mean ± SD, n = 2–7. MFI, mean fluorescence intensity. (I) Hematopoietic recovery after 5-fluorouracil (5-FU) challenge of WT or Esr1 / mice previously treated with tamoxifen or vehicle for 4 weeks. Values are means ± SD, n = 3–6. Data are representative of two independent experiments. In (F), (H), and (I), *p < 0.05, **p < 0.01 (vehicle versus tamoxifen). See also Figures S3 and S4.

tamoxifen treatment prevented the characteristic fibrosis and osteosclerosis of the BM associated with advanced disease (Figure 4D). Although—as expected from its bone anabolic effects—tamoxifen increased cortical bone thickness in both WT

(data not shown) and JAK2V617F+ mice (Figure 4D), tamoxifen prevented excessive bone formation inside the BM. To further investigate therapeutic benefits, mice with advanced disease (1,806 ± 542 3 103 platelets/ml) were administered



Figure 4. Tamoxifen Treatment Blocks the Progression of Myeloproliferative Neoplasms in Mice (A) Hematological parameters over time in peripheral blood from mice transplanted with Mx1-cre;JAK2V617F or control BM cells, treated with tamoxifen or vehicle (Figure S4A). Dots represent the mean. n = 8–10 (until week 12) or n = 4 (from week 16). Data are representative of two independent experiments. (B and C) Representative images and weight of spleens from mice transplanted with JAK2V617F+ or control BM cells, sacrificed 13 weeks posttransplant (i.e., after 9 weeks of tamoxifen/vehicle treatment). (D) Reticulin fiber (top) and Van Gieson (bottom) stainings of BM sections from mice transplanted with Mx1-cre;JAK2V617F BM cells, sacrificed 34 weeks posttransplant (i.e., after 30 weeks of treatment). Results are representative of n = 3–5 animals/group. Magnification: 2003 (top), 1003 (bottom). (E) Hematological parameters in peripheral blood of mice transplanted with Mx1-cre;JAK2V617F or control (WT) BM cells, treated with tamoxifen/vehicle for 4 weeks at thrombocytosis stage (9 weeks after transplantation). Tamoxifen doses (mg/kg/day, 3 times/week) are indicated. In (C and E), horizontal lines represent the mean. *p < 0.05, **p < 0.01, ***p < 0.001 (vehicle versus tamoxifen). See also Figure S5.

tamoxifen for 4 weeks. Treatment significantly reduced leukocyte and platelet counts and delayed the decrease in red cell parameters in peripheral blood characteristically associated with disease progression (Figure 4E and data not shown). Treatment of thrombocytotic mice with a 10-fold lower dose of tamoxifen,

comparable to the one chronically administered to healthy women in breast cancer prevention regimens, caused similar reductions in leukocyte and platelet counts and maintained normal red cell parameters (Figure 4E). Low-dose tamoxifen treatment of disease-free control mice only caused very mild



Figure 5. Tamoxifen Prevents JAK2V617F+ HSPC Expansion by Restoring Normal Apoptosis Levels (A) Number of colony-forming units in BM and spleen, and (B) immunophenotypically defined BM HSPCs in mice treated as in Figure 4A and sacrificed 13 weeks posttransplant (i.e., after 9 weeks of treatment). BM data correspond to one tibia, one femur, and one iliac crest. n = 4. (C) Hematological parameters over time in secondary recipients of tamoxifen- or vehicle-treated JAK2V617F+ BM cells. Lethally irradiated, untreated C57BL/6J mice received 2 3 106 pooled BM cells from mice in (A) and (B). n = 3–7. (D) Fraction of apoptotic HSPCs in the BM of mice in (A) and (B). Data are means ± SD, n = 4. NA, not available due to the very low cell number. *p < 0.05, **p < 0.01, ***p < 0.001 (vehicle versus tamoxifen).

thrombocytopenia (904.8 ± 74 versus 1,146 ± 78 3 103 platelets/ ml) and neutropenia (0.60 ± 0.09 versus 0.92 ± 0.16 3 103 neutrophils/ml; tamoxifen versus vehicle). These results suggest that tamoxifen, at a similar dose used for the treatment of other diseases, might be useful to treat MPNs at various stages. Tamoxifen Prevents JAK2V617F+ HSPC Expansion by Restoring Normal Apoptosis Levels Since MPNs are driven by mutant HSCs, we asked whether tamoxifen-induced MPN blockade was due to its effects on mutated HSPCs. JAK2V617F+ HSPCs (LT-HSCs, ST-HSCs, MPPs, and lin c-kit+ Sca-1 ) expressed Esr1 mRNA at levels similar to their WT counterparts, while Esr2 expression was practically undetectable (data not shown). Remarkably, tamoxifen treatment decreased HSPC numbers in the BM of JAK2V617F+

mice to the normal values found in untreated controls and significantly decreased their splenic infiltration (Figures 5A–5B and S5G). Like in disease-free mice, MPPs were the most affected HSPC, whereas LT-HSCs were less susceptible (Figure 5B). Secondary recipient animals transplanted with BM cells from tamoxifen-treated MPN mice showed delayed disease development and consistently low leukocyte/platelet counts, suggesting at least a partial elimination of MPN-initiating cells (Figure 5C). We next investigated whether these effects were caused by changes in cell death or proliferation. Tamoxifen modestly inhibited JAK2V617F+ HSPC cycle progression (Figure S5H). JAK2V617F confers a survival advantage to HSPCs that favors their accumulation (Kralovics et al., 2005). While mutant HSPCs exhibited comparatively lower apoptosis than their WT counterparts, tamoxifen increased the apoptotic fraction of every mutant



Figure 6. JAK2V617F+ HSPCs Are More Susceptible to Estrogens than Normal HSPCs (A–C) Contribution of the JAK2V617F+ clone to different hematopoietic lineages, measured as GFP chimerism in (A) peripheral blood, (B) BM and spleen, and (C) BM HSPCs of C57BL/6J mice competitively transplanted with equal numbers of WT and Mx1-cre;JAK2V617F;Rosa-gfp BM cells and treated with tamoxifen/ vehicle for 100 days. (D) Human leukocyte chimerism in peripheral blood of NSG mice transplanted with human CD34+ cells from JAK2V617F+ MPN patients and treated with tamoxifen/ vehicle for 4 weeks. (E) Number of hematopoietic (hCD45+) and myeloid (hCD45+ hCD33+) human cells (per two tibiae, one femur, and two iliac crests), and (F) fraction of apoptotic (Annexin V+) hCD45+ cells in the BM of mice in (D), analyzed after 5 months of treatment. n = 8–10. In (A), (C), and (F), data are means ± SD. In (B), (D), and (E), horizontal lines represent the mean. *p < 0.05, **p < 0.01. See also Figure S6.

HSPC subset to WT levels (Figure 5D). Thus, tamoxifen can block MPN progression by abrogating the survival advantage of mutated HSPCs and normalizing their numbers. JAK2V617F+ HSPCs Are More Susceptible to Estrogens than Normal HSPCs To compare the susceptibility of JAK2V617F+ and normal HSPCs to tamoxifen treatment, male mice were transplanted with equal numbers of BM cells from WT mice and Mx1-cre;JAK2V617F; Rosa-gfp mice and were treated with tamoxifen/vehicle for 100 days. These mice developed MPNs similar to recipients of

noncompetitive transplants, and tamoxifen also prevented their leukocytosis, thrombocytosis, erythrocytosis, and extramedular hematopoiesis, associated with reduced HSPC numbers in BM and spleen (Figures S6A–S6F). Most importantly, tamoxifen decreased the contribution of GFP+ (JAK2V617+) cells to the megakaryocytic lineage in blood, BM, and spleen, and reduced the mutant clone chimerism in circulating leukocytes (Figures 6A and 6B), indicating a more pronounced effect on mutant HSPCs. This effect was not observed in the erythroid lineage, consistent with the unaltered number of erythroid progenitors in the BM of treated animals (Figure S6F). Tamoxifen also decreased the



proportion of mutant HSPCs in the BM (Figure 6C). These results demonstrate that tamoxifen preferentially targets JAK2V617F+ HSPCs and facilitates hematopoietic recovery from normal HSPCs. To further assess the safety of tamoxifen treatment, we monitored mice transplanted with WT cells and treated with tamoxifen for 27 weeks. After this prolonged treatment, tamoxifen-treated mice showed virtually normal blood counts (only platelets were significantly decreased, but remained within normal values; Figure S6G). Interruption of tamoxifen treatment for 4 weeks normalized neutrophil and platelet counts (Figure S6H). Thus, tamoxifen treatment does not have significant detrimental effects on normal hematopoiesis. Tamoxifen Causes In Vivo Apoptosis of Human JAK2V617F+ HSPCs in a Xenograft Model To investigate the therapeutic effect of tamoxifen using an in vivo model of the human disease, CD34+ cells from JAK2V617F+ MPN patients were transplanted into immunodeficient NOD scid gamma (NSG) mice. Upon stable engraftment of the cells, mice were kept under tamoxifen or regular diet for 3 months. Tamoxifen significantly decreased the human cell chimerism in peripheral blood already after 4 weeks of treatment (Figure 6D), consistent with reduced number of human hematopoietic and myeloid-lineage cells in the BM (Figure 6E). This was associated with significant induction of apoptosis (Figure 6F), thus recapitulating the results obtained with the murine MPN model. Together, the data support an antineoplastic action of tamoxifen in both mouse and human MPNs, mediated by a proapoptotic effect on malignant HSPCs. Tamoxifen Enhances the Effect of Chemotherapy in MLL-AF9-Induced Acute Myeloid Leukemia To investigate the potential therapeutic targeting of ER signaling in a different hematological malignancy, we tested the ability of tamoxifen to sensitize chemoresistant MLL-AF9-induced acute myeloid leukemias (AMLs) to conventional chemotherapy. Mice transplanted with rtTA;MLL-AF9 BM cells received different combination regimens of tamoxifen and/or chemotherapy (Figure S7A). Shortly after chemotherapy, tamoxifen- and vehicletreated mice had similar numbers of MLL-AF9+ cells and HSPCs (Figures S7B–S7D). However, the reappearance of circulating leukemic cells after chemotherapy was delayed in tamoxifentreated mice (Figure 7A). Although the aggressiveness of this disease model led to similar lethality (Figure S7E), tamoxifen-treated mice showed fewer leukemic cells in BM, spleen, and blood (Figure 7B). Both 4OHT and E2 directly induced apoptosis of murine MLL-AF9+ blasts in vitro (Figure 7C) and 4OHT enhanced the proapoptotic effect of low-dose cytarabine on these cells (Figure S7F), suggesting a direct proapoptotic action of tamoxifen on leukemic cells. In vitro 4OHT treatment also induced apoptosis of human MLL-AF9+ AML cells (Figure 7D). Since estrogens are metabolic regulators and many tamoxifen-induced gene expression changes were related to metabolic pathways, we studied whether tamoxifen might compromise leukemic blast metabolism. Estrogen-induced apoptosis of MLL-AF9+ cells was preceded by a rapid, dose-dependent inhibition of mitochondrial respiration: both 4OHT and E2 rapidly decreased the baseline oxygen consumption rate and the spare

respiratory capacity. This metabolic effect was specific and reversible because the maximal respiratory capacity and the glycolytic flux (ECAR) remained unaltered (Figure 7E). These results show that tamoxifen can induce apoptosis of murine and human AML cells and can also sensitize leukemic cells to conventional chemotherapy. DISCUSSION Despite the known effects of estrogens on other cell types and their potential impact on the HSC niche through control of bone metabolism, whether sex hormones play a direct role in the regulation of primitive normal and leukemic hematopoietic cells has remained unclear. Estrogens have been shown to increase, decrease, or not affect HSPC numbers, depending on the methodology/markers used to define the cell populations (Illing et al., 2012; Jilka et al., 1995; Nakada et al., 2014; Thurmond et al., 2000). On the other hand, epidemiological data consistently show higher incidences of hematological malignancies (both lymphoid and myeloid) in men compared to women (American Cancer Society, 2013; Cancer Research UK, 2013), strongly suggesting a role for sex hormones in hematological cancer predisposition, and therefore in the regulation of HSPCs, from which these neoplasias arise. Here we described high-level expression of ERa in HSPCs and differential effects of ERa activation on HSPC proliferation and survival. While the more proliferative MPPs were most prone to apoptosis, the relatively quiescent LT-HSCs entered the cell cycle upon ER activation, resulting in loss of both self-renewal signatures and the ability to reconstitute hematopoiesis after stress (which requires activation of quiescent HSCs). The effects of tamoxifen were dependent on ERa activation in HSPCs and were cell autonomous, because they were abolished in Esr1 / HSPCs in vivo, even in a WT microenvironment. Increased expression of E2-induced genes by tamoxifen suggests that it acts as an ER agonist in HSPCs, as opposed to its antiestrogenic action on breast cancer cells. ER activation in LT-HSCs was accompanied by activation of the Myc transcriptional program, previously associated with HSC differentiation and consistent with the effects on self-renewal and poststress recovery. Myc is a transcriptional target of estrogens (Dubik and Shiu, 1988) and the effects of tamoxifen on hematopoietic progenitors seem at least partially dependent on Myc. Our findings are consistent with recent observations that HSCs divide more frequently in females and under conditions of high estrogen levels (Nakada et al., 2014); however, our data suggest more diverse effects of estrogen signaling on HSPC survival, proliferation, self-renewal, Myc activation, and stress response, and they demonstrate differential effects in various HSPC subsets. Based on the modulatory effects of ER activation on normal HSPC survival, quiescence, and self-renewal, we hypothesized that this pathway might exhibit therapeutic potential in hematological neoplasias. Indeed, tamoxifen alone blocked the development of JAK2V617F+ MPNs by reestablishing normal apoptosis levels in malignant HSPCs, which are otherwise protected from cell death. Notably, tamoxifen could also rescue leukocytosis and thrombocytosis when administered at an advanced disease stage, at a similar dose as the one used clinically for breast cancer prevention.



Figure 7. Tamoxifen Reduces Leukemic Burden in a Mouse Model of MLL-AF9-Induced Acute Myeloid Leukemia (A) Leukocyte counts over time in peripheral blood from C57BL/6J mice transplanted with rtTA;MLL-AF9;CD45.1 and WT BM cells, treated as in Figure S7A. Values indicate mean ± SEM. n = 5–7 (until day 32), n = 3 (from day 41). *p < 0.05 (chemo versus TAM+chemo). Results are representative of two independent experiments. (B) Leukemic burden in BM, spleen, and blood of mice treated as in (A), sacrificed when moribund. Data are means ± SD. n = 3–9. Chemo, chemotherapy; ++ indicates the ‘‘intensive’’ regimen. *p < 0.05. Data were pooled from two independent experiments. (C and D) Fraction of apoptotic mouse (C) and human (D) MLL-AF9+ BM blasts treated in vitro with 4OHT or E2 for 48 hr. Data indicate mean ± SD of triplicate measurements. (E) Baseline and maximal respiratory capacity, expressed as oxygen consumption rate (OCR); spare respiratory capacity (SRC); and extracellular acidification rate (ECAR) in murine MLL-AF9+ BM blasts treated with the indicated concentrations of 4OHT or E2 for 2 hr. Data are means ± SD of five or six replicates (one representative experiment out of three is shown). In (C)–(E), *p < 0.05, **p < 0.01 (treated versus untreated). See also Figure S7.

Although tamoxifen did not exclusively target malignant HSPCs, we provided multiple evidences demonstrating higher susceptibility of JAK2V617F+ HSPCs: (1) reduction of the mutant clone chimerism in competitive transplantation experiments; (2) differential effects on extramedullary HSPC infiltration (elimination of splenic HSPCs in MPN-affected mice versus HSPC mobilization to the spleen in control mice); (3) higher apoptosis in JAK2V617F+ LT-HSCs than in their normal counterparts; and (4) virtually normal hematological parameters in tamoxifen-treated WT mice, with only mild, reversible thrombocytopenia/neutropenia. Similar mild side effects were noted since the initial breast cancer trials (Gong et al., 2011; Lerner et al., 1976), but the positive efficacy/safety profile of tamoxifen has allowed us to extend

its use from breast cancer treatment to prevention (Cuzick et al., 2013). The above observations argue for the feasibility of using tamoxifen in MPN therapy with acceptable safety and relatively low toxicity. In addition, tamoxifen enhanced the response of MLL-AF9induced AMLs to conventional chemotherapy in vivo, reducing leukemic burden despite the aggressive course of the disease. Tamoxifen induced apoptosis of murine and human MLL-AF9+ cells, preceded by rapid inhibition of mitochondrial respiration. Hormone replacement therapy has been previously associated with a slightly decreased risk of developing AML (Ross et al., 2005). Esr1 gene polymorphisms have been suggested to confer AML susceptibility (Anghel et al., 2010). The Esr1



gene is frequently methylated in AML (Agrawal et al., 2007; Herman et al., 1996; Hess et al., 2008), its methylation status might predict prognosis (Yao et al., 2010), and its re-expression after hypomethylating therapy correlates with clinical response (Blum et al., 2007). Finally, it is important to note the wide use of tamoxifen in experimental research to induce CreER recombinase activity. The effects of tamoxifen on HSPCs have probably remained underappreciated due to its relatively low impact in normal blood counts. However, our data call for caution when interpreting results, since even in the absence of any genetic modification, tamoxifentreated animals exhibit altered baseline HSPC populations and may show, depending on the experimental conditions, some other abnormal hematological parameters. A previous study has raised similar concerns about polyI-polyC-inducible models due to the proliferative effect of interferon-a on HSCs (Essers et al., 2009). Together, these data reveal differential roles of estrogen signaling in the regulation of primitive normal and malignant hematopoietic cells. They also demonstrate that targeting this pathway with a clinically approved ligand, at doses with proven acceptable toxicity profiles in humans, provides a potential therapeutic strategy for a set of neoplasias currently without definitive cure. EXPERIMENTAL PROCEDURES Mice and Pharmacological Treatments All mouse procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 53/2013. Animals were housed in specified-pathogen-free facilities. Age-matched, WT C57BL/6J (CD45.2+), B6.SJL (CD45.1+), Esr1 / and Esr2 / (Jackson Laboratory), Pol2-creERT2 (Guerra et al., 2003), Myc-floxed (de Alboran et al., 2001), and NSG mice were used for in vivo treatments and as donor/recipients in BM transplantation assays. MPN was induced by transplantation of 2 3 106 polyIpolyC-induced Mx-Cre;JAK2V617F BM cells into lethally irradiated C57BL/6J recipients. AML was modeled by transplantation of 106 rtTA;MLL-AF9 BM cells followed by doxycycline administration. For short treatments (up to 1 month), mice were injected i.p. with 14 or 140 mg/kg of tamoxifen or vehicle (corn oil), three times per week on alternate days. For longer treatments, mice were fed with tamoxifen-containing diet TM400 (Harlan) after the last of four to six i.p. injections. Competitive Repopulation Assays Recipient mice (C57BL/6J or B6.SJL) were lethally irradiated (137Cs source, 12 Gy whole-body irradiation, split dose 6.0 + 6.0 Gy, 3 hr apart) prior to BM transplant. For AML/MPN experiments, recipients received transplants as described above. For competitive repopulation assays, CD45.2+ BM cells were transplanted into CD45.1+ recipient mice or vice versa, mixed with competitor BM cells isogenic to the recipient (typically 2 3 105 cells; see Figure Legends for details). Peripheral blood chimerism was assessed by flow cytometry every 2–4 weeks, and recipients were sacrificed 4 months posttransplant for the analysis of long-term BM engraftment. The models of hematological neoplasia and the methods for hematopoietic recovery after myelotoxic stress, flow cytometry and fluorescence-activated cell sorting, hematopoietic progenitor assays and other in vitro assays, RNA sequencing and bioinformatic data analysis, western blot, and histological and statistical analysis are described in detail in the Supplemental Experimental Procedures.

SUPPLEMENTAL INFORMATION Supplemental Information for this article includes seven figures, three tables, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.stem.2014.11.002. ACKNOWLEDGMENTS We thank S.B.C. Lama, A.M. Martıń de Ana, A.B. Ricote, J.M. Ligos, S. Bartlett, and the CNIC Genomics and Bioinformatics Units for assistance; M. Torres and C. Claverıá for their kind gift of Pol2-creERT2;MycloxP/loxP mice; members of S.M.-F. and M. Ricote labs for helpful discussions; and G. Roncador and A. Kerguelen for kindly providing reagents. This work was supported by the Centro Nacional de Investigaciones Cardiovasculares Carlos III; the Spanish Ministry of Economy and Competitiveness (Plan Nacional grant SAF-201130308 to S.M.-F.; Ramoń y Cajal Program grants RYC-2011-09726 to A.S.A., RYC-2011-09209 to J.I., and RYC-2009-04703 to S.M.-F.; Spanish Cell Therapy Network TerCel to S.M.-F.); Marie Curie Career Integration Program grants (FP7-PEOPLE-2011-RG-294262/294096) to A.S.-A. and S.M.-F.; and a ConSEPOC-Comunidad de Madrid grant (S2010/BMD-2542) to S.M.-F. A.G.-G. is the recipient of Fundacioń Ramoń Areces and Fundacioń La Caixa fellowships. This research was partly funded by a European Hematology Association Research Fellowship awarded to A.S.-A. S.M.-F. is supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. Received: July 18, 2014 Revised: October 8, 2014 Accepted: November 5, 2014 Published: December 4, 2014 REFERENCES Adkins, D., Brown, R., Trinkaus, K., Maziarz, R., Luedke, S., Freytes, C., Needles, B., Wienski, D., Fracasso, P., Pluard, T., et al. (1999). Outcomes of high-dose chemotherapy and autologous stem-cell transplantation in stage IIIB inflammatory breast cancer. J. Clin. Oncol. 17, 2006–2014. Agrawal, S., Unterberg, M., Koschmieder, S., zur Stadt, U., Brunnberg, U., Verbeek, W., Bu¨chner, T., Berdel, W.E., Serve, H., and Mu¨ller-Tidow, C. (2007). DNA methylation of tumor suppressor genes in clinical remission predicts the relapse risk in acute myeloid leukemia. Cancer Res. 67, 1370–1377. American Cancer Society (2013). Cancer Facts & Figures. http://www.cancer. org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-figures2013. Anghel, A., Narita, D., Seclaman, E., Popovici, E., Anghel, M., and Tamas, L. (2010). Estrogen receptor alpha polymorphisms and the risk of malignancies. Pathol. Oncol. Res. 16, 485–496. Arranz, L., Herrera-Merchan, A., Ligos, J.M., de Molina, A., Dominguez, O., and Gonzalez, S. (2012). Bmi1 is critical to prevent Ikaros-mediated lymphoid priming in hematopoietic stem cells. Cell Cycle 11, 65–78. Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., et al.; Cancer Genome Project (2005). Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365, 1054–1061. Blum, W., Klisovic, R.B., Hackanson, B., Liu, Z., Liu, S., Devine, H., Vukosavljevic, T., Huynh, L., Lozanski, G., Kefauver, C., et al. (2007). Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia. J. Clin. Oncol. 25, 3884–3891.

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Molecular diagnosis of hematopoietic neoplasms.

PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia.

Targeting prohibitins induces apoptosis in acute myeloid leukemia cells.

Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes.

Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Globalization, Volunteerism, and Mission Surgery: How to Help Without Harming.

Deferasirox improves hematopoiesis after allogeneic hematopoietic SCT.

Subsequent malignant neoplasms in pediatric cancer patients treated with and without hematopoietic SCT.

Resveratrol prevents rapamycin-induced upregulation of autophagy and selectively induces apoptosis in TSC2-deficient cells.

Extramedullary Manifestations of Myeloid Neoplasms.

Xanthohumol inhibits Notch signaling and induces apoptosis in hepatocellular carcinoma.

Isocitrate dehydrogenase (IDH)2 R140Q mutation induces myeloid and lymphoid neoplasms in mice.

BRCC3 mutations in myeloid neoplasms.

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

Heterogeneity of clonal expansion and maturation-linked mutation acquisition in hematopoietic progenitors in human acute myeloid leukemia.

progenitor cells and hematopoiesis.

T-cell-restricted T-bet overexpression induces aberrant hematopoiesis of myeloid cells and impairs function of macrophages in the lung.

Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages.

Fish Oil-Rich Diet Promotes Hematopoiesis and Alters Hematopoietic Niche.

Thioredoxin-interacting protein, hematopoietic stem cells, and hematopoiesis.

The Cebpa +37-kb enhancer directs transgene expression to myeloid progenitors and to long-term hematopoietic stem cells.

Jak2a regulates erythroid and myeloid hematopoiesis during zebrafish embryogenesis.

A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

SH003 selectively induces p73‑dependent apoptosis in triple‑negative breast cancer cells.