Treatment With Hedgehog Inhibitor, PF-04449913, Attenuates Leukemia-Initiation Potential In Acute Myeloid Leukemia Cells

Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell (LSC) populations in several experimental systems. Cumulative evidence suggests that dormant self-renewing LSC contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. PF-04449913 (PF) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF has shown promising Results regarding safety, tolerability, and early signs of efficacy in a phase 1 dose-escalation study of hematologic malignancies including AML (Jamieson C, et al. ASH, 2011). On the basis of these encouraging Results, phase 1b and phase 2 studies of PF in combination with chemotherapies have been planned in patients with AML. However, the detailed mechanisms and biomarkers remain to be elucidated in AML therapy with Hh pathway inhibitors.

We used AML cell lines and patient-derived primary AML cells in order to evaluate the efficacy and elucidate detailed mechanisms and biomarkers in the Hh antagonist, PF treatment. Using the co-culturing system with HS-5 stromal cells, the colony assay system, and the immunodeficient NOD/SCID/IL2rg^null (NOG) mouse model serially xenotransplanted with primary AML cells, we examined the effects of PF on LSC population and AML propagation.

Using FACS sorting and RQ-PCR assays of AML patient-derived primary cells, the Hh signaling pathway was activated more in CD34-positive cells than CD34-negative cells. Ex vivo-treatment with PF inhibited proliferation and induced minimal cell death in leukemia cell lines and primary AML cells. However, in vivo-treatment with PF attenuated leukemia-initiation potential in AML cells through the serial transplantation system, while limiting reduction of tumor burden in the primary leukemia system. Also in the colony-assay system using primary AML cells, treatment with PF reduced serially colony formation. In MOLM-14 cells, treatment with PF down-regulated mRNA encoding downstream effector GLIs and GLI-targeting molecules in the canonical Hh pathway using RQ-PCR assays, and decreased nuclear expression of GLI-2 using immunofluorescence assays. In addition, treatment with PF remarkably decreased the quiescent (Hoechst-33342^low/Pyronin-Y^low) cell population and increased cycling cell population. In the in vivo-NOG mouse system, comprehensive Gene Set Enrichment Analysis (GSEA) revealed that PF treatment modulated cell cycle regulation and self-renewal signaling in primary AML cells. Moreover, combined treatment with PF abrogated resistance to Ara-C in AML cell lines co-cultured with HS-5 stromal cells and sensitized primary AML cells to Ara-C in the colony-assay system. We are also investigating toxicity for normal cord blood cells with PF treatment.

Our findings imply that selective Hh inhibitor, PF treatment can attenuate the leukemia-initiation potential in AML cells by modulation of cell cycle regulation and self-renewal signaling, and can also improve AML therapy through sensitizing dormant LSC to chemotherapy and overcoming the resistance in the bone marrow microenvironment.

Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding.