A PML-PPAR pathway for fatty acid oxidation regulates ...

Nature Medicine doi:10.1038/nm.2882

A PML-PPAR pathway for fatty acid oxidation regulates haematopoietic stem cell maintenance

Keisuke Ito1,7*, Arkaitz Carracedo1,8*, Dror Weiss1, Fumio Arai2, Ugo Ala1, David E. Avigan3, Zachary T. Schafer4, Ronald M. Evans5, Toshio Suda2, Chih-Hao Lee6 & Pier Paolo Pandolfi1,9

1Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Division of Genetics, Departments of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 2Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan 3Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 4Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 5Gene Expression Laboratory, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92037, USA 6Department of Genetics and Complex Diseases, Division of Biological Sciences, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA 7Current addresses: Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Departments of Cell Biology, Stem Cell Institute and Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA 8Current addresses: CIC bioGUNE, Technology Park of Bizkaia, 48160 Derio, Bizkaia, Spain; IKERBASQUE, Basque Foundation for Science

9Correspondence to: P.P.P.: ppandolf@bidmc.harvard.edu * These authors contributed equally to this work

Supplementary Figure 1-12 and legends Supplementary Table 1-2

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Nature Medicine doi:10.1038/nm.2882

Supplementary Figure Legends Supplementary Figure S1. Functional analysis of Ppard-deleted HSCs. (a) Relative expression of Ppara, Ppard and Pparg in CD34negKSL cells. Data from a representative experiment is shown (mean ? s.d., out of 3 independent experiments). Mouse liver tissue is presented as a control. (b) Reduction of Ppard levels after transduction with Cre-expressing retrovirus in stem cell population. KSL cells from PpardF/F mice were infected with MSCV-Puro-ires-GFP (Empty) or MSCV-Puro-Cre-ires-GFP (Cre) and mRNA was extracted from infected CD34negKSL cells. Gene expression of Ppara, Ppard and Pparg was analyzed by qRT-PCR using specific primers. U.D., undetectable. (c) Normal homing capacity to the bone marrow (BM) of Ppard-depleted HSC. Lethally irradiated Ly45.1 mice were transplanted with 3,000 KSL cells from PpardF/F mice (Ly45.2) infected with Empty or Cre vector and 2x105 competitor BMMNCs. Cells from the BM of recipient mice were analyzed 24 hours posttransplantation to identify their derivation. The mean percentages ? s.d. of donor-derived KSL cells are shown (n = 3). Representative flow cytometry data are shown in insets. (d) Haematopoietic reconstitution capacity in bone marrow transplantation (BMT) of Cre-expressing Ppard+/+ and PpardF/F. Haematopoietic contribution of donor-derived cells at the indicated number of weeks after BMT is shown (n = 4). (e) Gene expression profile in purified CD34negKSL cells infected with Empty or Cre vector. Relative gene expression compared with CD34negKSL cells infected Empty vector is shown. (f) Acute loss of Ppard results in reduced cobblestone area forming cells (CAFC) in HSCs. CD34negKSL cells transduced with Empty or Cre were purified and their CAFC frequencies in the limiting-dilution assay (LDA) were determined using Poisson statistics. In each experiment, 36 replicates of each dilution were performed (n = 4). (g) Decreased long-term colony forming capacity in Ppard-depleted HSCs. KSL cells infected with Empty or Cre vector were cultured with stromal cells for the indicated number of weeks and tested for colony formation (n = 3). (h) Reduced colony

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Nature Medicine doi:10.1038/nm.2882

forming capacity in Ppard-depleted HSCs in long-term culture initiating cell (LTC-IC) limiting dilution assay. CD34negKSL cells transduced with Empty or Cre were purified and their LTC-IC frequencies in the limiting dilution assay (LDA) were determined using Poisson statistics. In each experiment, 12 replicates of each dilution were performed (n = 4). (i) Comparison of LTC-IC frequencies in LDA between Cre-expressing Ppard+/+ and PpardF/F. (j) Decreased ATP levels in Ppard-deleted HSCs. ATP levels were determined in PpardF/F CD34negKSL cells infected with Empty or Cre vector. (k) No significant effect of Ppard-ablation or Cre expression was observed in progenitor proliferation. Colony forming unit in culture (CFU-C) of PpardF/F infected with Cre vector or Ppard+/+ KSL infected with Empty or Cre vector were investigated (k, left). Differentiation was assayed using single-cell liquid culture of those KSL (k, right). (l) Cell cycle status of Ppard-deleted HSCs. Lethally irradiated Ly45.1 mice were transplanted with 2,000 CD34negKSL cells from PpardF/F mice (Ly45.2) infected with Empty or Cre vector and 2x105 competitor BMMNCs. 6 weeks after BMT, intracellular Ki67 negative cells were determined in donor-derived CD150posCD48negKSL cells (n = 4). All error bars indicate s.d.

Supplementary Figure S2. Pharmacological PPAR activators enhance HSC maintenance. (a-d) As shown in (a), mice were treated with GW for 4 weeks, and progenitor function (b), progenitor compartment (c) and long-term culture initiating cell (LTC-IC) capacity (d) were analysed. Colony forming unit in culture (CFU-C) of KSL cells treated with vehicle (Ctrl) or GW was investigated (b, left). Differentiation was assayed using single-cell liquid culture of KSL cells as previously described (b, right). GW treatment did not affect progenitors numbers (c). Results are mean relative numbers ? s.d. of Linneg (Linneg), GMP (granulomyeloid progenitor; LinnegSca-1negc-KitposIL7RnegCD34posCD16/32pos), MEP (megakaryo-erythrocytic progenitors LinnegSca-1negc-KitposIL7RnegCD34negCD16/32neg), CMPs (common myeloid progenitors; LinnegSca-1negc-

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Nature Medicine doi:10.1038/nm.2882

KitposCD34posCD16/32low) and CLPs (common lymphoid progenitors; LinnegSca-1loc-KitloIL-7Rpos) in mice treated with vehicle (Ctrl) or GW. LTC-IC frequencies in KSL cells treated with GW were determined by LDA using Poisson statistics (n = 4) (d). In each experiment, 12 replicates of each dilution were performed (n = 4). (e) PPAR activators increase cobblestone area forming capacity (CAFC) in HSCs in limiting dilution assay (LDA). In each experiment, 36 replicates of each dilution were performed (n = 4). (f) GW enhances PPAR signalling in HSCs. Levels of Ppard targets Cpt1a and Angptl4 in CD34negKSL cells treated overnight with GW were measured by q-RT-PCR. Data shows relative expression ? s.e. (n = 4). Data is corrected by RpLp0 (36B4) (left) as well as by -actin (right). Cpt1a, carnitine palmitoyltransferase 1. Angptl4, Angiopoietin-related protein 4. (g) GW increases ATP levels in the HSC. ATP levels were determined in CD34negKSL cells treated with GW (n = 3). (h) Treatment with GW501516 (GW; red) or L165,041 (L165; blue) in vitro increases colony formation of HSCs in long-term culture initiating cell (LTC-IC) assay. CD34negKSL cells were cultured with stromal cells for the indicated number of weeks (W) with GW or L165 and tested for colony formation, (n = 3). (i) GW increases long-term culture initiating cells (LTC-IC) in LDA assay (n = 4). (j) GW increases CD34negKSL compartment. After transplantation, recipient mice were treated with vehicle or GW for 4 weeks and were then observed for 2 weeks without treatment. Representative flow cytometry data are also shown in insets. (k,l) GW increases the long-term HSC compartment. After transplantation, recipient mice were treated with vehicle or GW for 2 weeks and were then observed for 8 weeks without treatment. All error bars indicate s.d. unless indicated otherwise.

Supplementary Figure S3. PPAR agonist GW increases the maintenance of HSCs in a Pparddependent manner. GW treatment did not affect CAFC (limiting dilution assay (LDA, a), LTC-IC capacity (limiting dilution assay, b), and repopulation capacity of BM mononuclear cells (limiting dilution assay, c) in Ppard-depleted HSCs. All error bars indicate s.d.

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Nature Medicine doi:10.1038/nm.2882

Supplementary Figure S4. Modulation of PPAR signalling in vitro. Sorted KSL cells from 8-week old mice cells were culture in retronectin-coated well supplemented with SCF and TPO. Cells were either maintained untreated or treated with vehicle, 0.1 M GW501516 (Enzo life sciences) or 10 M Etomoxir (Sigma) (a). After 3 days co-culture, CD34negKSL cells were sorted and frequencies of cobblestone area forming cells (CAFC, b) and long-term culture initiating cells (LTC-IC, c) were determined in the limiting dilution assay (LDA) using Poisson statistics (n = 4).

Supplementary Figure S5. Etomoxir treatment reduces HSC maintenance without induction of apoptosis. (a) Significant reduction of Ppar signalling in the differentiated compartment. Levels of Cpt1a, Acox1 and Adrp were measured by q-RT-PCR, using -actin as endogenous control. Data shows relative expression ? s.e. in the fractionated populations normalized over the expression in CD34negKSL cells (n = 10). (b) Etomoxir inhibits maintenance of long-term HSC population. After transplantation, recipient mice were treated with vehicle or Etomoxir for 2 weeks. Mean numbers ? s.d. of donor-derived CD34negFlt3negKSL cells 10 weeks after transplantation in recipient mice are shown. (c,d) KSL cells were co-cultured with stromal cells (c) or transplanted into irradiated recipient mice (d) and treated with Etomoxir for 2 weeks. Relative number of CD34negKSL cells (c, left), total number of CD34negKSL cells (d, left) and apoptosis in CD34negKSL cells (c,d, right) were analysed (n = 3). (e) The dose of Etomoxir employed in vivo did not have toxic effect. 8-week old WT mice were treated with Etomoxir (25 mg per kg body weight, day ON day OFF) for 4 weeks. BW is checked every 2 weeks (n = 3) (left). 4 weeks after Etomoxir treatment (Tx), organ weight was compared with vehicle treated mice (Ctrl) (n = 3). (f) Pharmacological inhibition of mitochondrial FAO with Etomoxir decreases cobblestone area forming cells (CAFC) in the HSC compartment. CAFC frequencies in CD34negKSL cells treated Etomoxir were determined by limiting-dilution assay (LDA)

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