Background: Cumulative evidence suggests that dormant self-renewing leukemic stem cells (LSCs) contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. Therefore, there is a great need to find innovative therapies for AML that eliminate LSCs by targeting their specific properties. Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in maintenance of the LSC populations in several experimental systems. PF-0444913 (PF-913) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF-913 has shown promising results regarding safety, tolerability, and early signs of efficacy in the early phase study of hematologic malignancies, including AML (Jamieson, et al. ASH, 2011). However, the detailed mode of action and biomarkers remain to be elucidated in AML therapy with the Hh pathway inhibitors.

Methods: We used AML cell lines and patient-derived primary AML cells and bone marrow cells in PF-913 treatment. Using RQ-PCR assays, we examined the change of gene expressions in the canonical Hh pathway molecules. We carried out comprehensive gene set enrichment analysis (GSEA), and pathway analysis during PF-913 treatment both in the pre-clinical experimental systems and the clinical setting (bone marrow blast-rich mononuclear cells derived from AML patients during the PF-913 mono-therapy). We also examined cell cycle analyses using flow-cytometory and Ki-67 immuno-staining assays.

Results: In primary AML cells, the Hh signaling pathway was activated more in CD34+ cells than in CD34- cells. In vitro treatment with PF-913 induced a minimal cell death-accompanied decrease of the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population. Through the serial transplantation immunodeficient NOD/SCID/IL2rgnull (NOG) mouse model xenotransplanted with primary AML cells, in vivo treatment with PF-913 attenuated leukemia initiation potential in AML cells (0% human-CD45+ cells in bone marrow cells derived from the 2nd recipient mice), while limiting reduction of tumor burden in the primary xenotransplanted recipient mice. In the NOG mouse model and the ex vivo culturing system of AML cell lines, GSEA revealed that PF-913 treatment induced effects on the self-renewal signatures and the cell-cycling regulations associated with LSC-like properties. Moreover, GSEA revealed that PF-913 treatment in the clinical setting modulated the LSC-like signature, the cell-cycling regulation signature, and the chemokine activity signature in the AML bone marrow cells. Ki-67 immuno-staining of bone marrow derived from AML patients showed that PF-913 treatment temporarily increased cell-cycling status during shorter periods of treatment. We also examined the pluripotency factor, Nanog expression in bone marrow cells derived from AML patients during the PF-913 therapy, based on the previous report that downstream effectors in the Hh pathway, GLI1 and GLI2, directly bind to the Nanog promoter and that the GLI-Nanog axis promotes stemness and growth in several cancers. Change of Nanog transcripts was closely associated with the GLI-target genes in bone marrow blast-rich mononuclear cells derived from AML patients during the PF-913 therapy. Furthermore, by backing to the pre-clinical experimental systems, we are investigating more detailed mechanisms of Nanog regulation during PF-913 treatment and more rationale methods of combination treatment with PF-913 and other candidate drugs (such as conventional chemo-drugs and other type of molecular targeted inhibitors).

Conclusion: Gene profiling analyses revealed that treatment with Hh signaling inhibitor, PF-913 modulates self-renewal and cell-cycling signatures in AML, and Nanog transcript can be a responsive biomarker during the therapy.Our findings imply that PF-913 treatment can improve AML therapy through sensitizing dormant LSCs to chemotherapy and overcoming residual LSC-like diseases in the bone marrow microenvironment.


Minami:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Naoe:Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Toyama Chemical CO., LTD.: Research Funding; Celgene K.K.: Research Funding. Minami:Novartis: Honoraria, Other: Member of IDMC , Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.