Valproic Acid Stimulates Proliferation and Self-renewal of Hematopoietic Stem Cells Gesine Bug, Hilal Gu ¨l, Kerstin Schwarz, Heike Pfeifer, Manuela Kampfmann, Xiaomin Zheng, Tim Beissert, Simone Boehrer, Dieter Hoelzer, Oliver Gerhard Ottmann, and Martin Ruthardt Medizinische Klinik II/Abteilung Ha ¨matologie, Klinikum der Johann Wolfgang Goethe-Universita ¨t, Frankfurt, Germany Abstract Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differ- entiation block in leukemic blasts, an effect achieved either alone or in combination with differentiating agents, such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompa- nied by a down-regulation of p21 cip-1/waf-1 . Furthermore, valproic acid inhibits GSK3B by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4 , a target gene of Wnt signaling. Both are known to directly stimulate the prolifer- ation of HSC and to expand the HSC pool. In summary, we here show that valproic acid, known to induce differentiation or apoptosis in leukemic blasts, stimulates the proliferation of normal HSC, an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. (Cancer Res 2005; 65(7): 2537-41) Introduction Acute myeloid leukemia (AML) is characterized by a differ- entiation block leading to the accumulation of immature blasts in the bone marrow. The vast majority of AML-patients are over 60 years old and experience a median survival below 1 year even if treated with intensive chemotherapy (1). Therefore, there is the necessity to develop alternative treatment strategies, such as a differentiation-inducing therapy employing histone deacetylase inhibitors (HDI). HDI have been shown to promote differentiation either alone or in combination with differentiating agents such as all-trans retinoic acid (t-RA; refs. 2, 3). Valproic acid (4), a potent HDI which has been safely used for over two decades in the therapy of epilepsy and bipolar disorders, is now under clinical evaluation (5). In a current clinical study we are evaluating a valproic acid/t-RA combination therapy on patients suffering from advanced myeloid malignancies (6). Patients responding to this therapy frequently developed constant or increased bone marrow cellularity despite a remarkable blast cell reduction and peripheral hypergranulocytosis (>100,000 cells/L; ref. 6). In one patient, it was possible to distinguish malignant from normal hematopoiesis by the presence of the isochromosome (17)(q10) in the leukemic blasts. The analysis revealed that whereas the CD34 + progenitor cells contained residual i(17)(q10), all granulocytes had a normal karyotype, suggesting dominance of normal hematopoiesis over the malignant clone. 1 Based on the hypothesis that this clinical picture might be related to a still unknown effect of valproic acid, we here investigated its activity on hematopoietic stem cells (HSC) with respect to differentiation, proliferation as well as to self-renewal. Materials and Methods Enrichment of human and murine hematopoietic stem cells. Bone marrow was obtained from healthy donors and umbilical cord blood was collected with informed consent of the donors or mothers, respectively. Isolation of mononuclear cells, CD34 + cell selection, and isolation of Sca1 + /lin À HSC from C57BL/6J (Ly5.2) female mice or congenic C57BL/6.SJL-Ly5.1 mice (Charles River, Sulzfeld, Germany) were done as previously described (7, 8). Culture and colony assay of CD34 + hematopoietic stem cells. CD34 + cells were cultured and colony formation of CD34 + HSC was assessed as previously described (7). Cells were harvested after 7 days, counted, and assayed for CD34 and CD14 expression as previously described (7). Replating efficiency and differentiation of Sca + /lin À hematopoietic stem cells. Colony formation, replating efficiency, and differentiation were assessed as recently described (8). Day 12 spleen colony-forming unit assay. After 2 days of culture, all cells that grew from 1,000 Ly5.2 Sca1 + /lin À cells were injected into lethally irradiated (10 Gy) female Ly5.1 recipients 8 to 12 weeks of age. Transplanted mice were euthanized 12 days later. Spleens were either fixed in Bouin’s fixative for 5 minutes, then transferred to 10% neutral buffered formalin (Sigma, Steinheim, Germany; ref. 9) or cells were prepared for surface marker analysis as described (8). Competitive repopulation assay. After 2 days of culture, all cells that grew in culture from 1,000 Ly5.2 Sca1 + /lin À cells under each culture condition were injected into lethally irradiated Ly5.1 female recipients 8 to 12 weeks together with 1 Â 10 5 normal Ly5.1 bone marrow cells (9). Transplanted mice were euthanized 12 weeks later and mononuclear bone marrow cells were isolated and stained with conjugated monoclonal antibodies specific for Ly5.2 and Ly5.1 or mouse IgG2a (all from PharMingen, San Diego, CA) for 30 minutes at 4jC for fluorescence- activated cell sorting analysis. Requests for reprints: Martin Ruthardt, Medizinische Klinik II/Abteilung Ha ¨matologie, Klinikum der Johann Wolfgang Goethe-Universita ¨t, Theodor-Stern Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-6301-5338; Fax: 49-69-6301-6131; E-mail: ruthardt@em.uni-frankfurt.de. #2005 American Association for Cancer Research. 1 Bug et al., submitted for publication. www.aacrjournals.org 2537 Cancer Res 2005; 65: (7). April 1, 2005 Priority Reports Research. on December 1, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from