FoxO3a is essential for survival of chronic myeloid leukemia-initiating cells Naka Kazuhito1, Noboru Motoyama2, Atsushi Hirao1 (1 Division of Molecular Genetics, Cancer Research Institute, Kanazawa University, 2 Department of Geriatric Medicine, National Institute for Longevity Sciences, National Center for Gerontology and Geriatrics) Chronic myelogenous leukemia (CML) is attributed to a defined genetic abnormality, BCR-ABL, a constitutively active protein tyrosine kinase. Although the introduction of imatinib, a small molecule Setup: inhibitor of ABL, represented a breakthrough in the treatment of CML, major part of patients treated Size: A4. Page therapy due to in chronic-phase CML are not offNumber: Single. resistance or intolerance. Recent studies have Orientation: Portrait. Margins: 25mm of the direction. suggested that imatinib is a potent inhibitorin everyproduction of differentiated leukemic cells, but Characters per line: 40 characters. does not deplete leukemic stem cells. To date, therapeutics that can eradicate CML-initiating cells, Lines per page: 38 lines. Character’s font: Times New Roman. Character’s size: 12 these however, have remained under investigation. To overcomepoints. clinical problems, we here evaluated molecular mechanisms on survival of the CML-initiating cells. We first generated a mouse Space: Single line spacing Lines order: CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature Title: bold , cells, and the cells hematopoietic aligned to the left. were subsequently transplanted into irradiated recipient mice. The Two blank lines. transplantation experiments showed that CML-initiating cells were highly enriched in Researchers’ names: aligned to the left with the presenter’s name underlined. c-Kit+Lin-Sca-1+ (KLS+) population in BCR-ABL+ CML cells, as previously described. Affiliation: aligned to the left and enclosed between brackets. Unexpectedly, phosphorylation level of Akt in KLS+ population as CML-initiating cells appeared to Two blank lines. be lower than that in non- the left cells, KLS- cells, despite it is widely believed that BCR-ABL Abstract text :aligned to initiating induces activation of Akt signal. Since Forkhead O (Foxo) transcription factors, which are important downstream targets of PI3K-Akt signaling, are essential for the maintenance of self-renewal capacity of normal HSCs, we focused on analysis of Foxo3a in CML. Consistent with Akt phosphorylation status, we found that approximately 40% of CML KLS+ showed nuclear localization of Foxo3a, whereas majority of KLS- cells showed the cytoplasmic localization, suggesting that Foxo3a is activated via Akt inactivation in the rare CML-initiating cells, but not in majority of CML cells. Serial transplantation experiments for CML-initiating cells originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency attenuated the ability of CML-initiating cells to develop leukemia at third round transplantation experiments. Number of Annexin-V+ cells was higher in the Foxo3a-deficient CML-initiating cells than those seen in wild-type CML-initiating cells in vivo. Furthermore, deficiency of Foxo3a led to enhanced efficiency in elimination of CML-initiating cells in combination with imatinib treatment. These results demonstrate a critical role of Foxo pathway in survival of drug-resistant CML-initiating cells, and provide a novel therapeutics approach for CML patients by suppression of FOXO signaling.