Chemical Inhibition of the Macrophage Inhibitor Factor (MIF) Induces Cell Cycle Arrest and Apoptosis in Lymphoid and Myeloid Leukemia

More selective and less toxic chemotherapeutic agents are needed as frontline treatments in high-risk patients and as salvage agents in relapse disease. In a search for natural products with anti-cancer activity, we previously reported that the isothiocyanate sulforaphane has anti-leukemic properties in pediatric T- and B-cell acute lymphoblastic leukemia by inducing G2/M cell cycle arrest and apoptosis (PLOS ONE, 2012). We hypothesized that sulforaphane forms adducts in leukemia cells resulting in the inactivation of proteins required for survival. Here we report that the macrophage inhibitor factor (MIF) is targeted by sulforaphane in lymphoid leukemia cells. Taking advantage of the electrophilic carbon of isothiocyanate (N=C=S) group in sulforaphane that spontaneously forms thionacyl adducts with sulfhydryl groups of cellular proteins, we performed a cell displacement assay by competition of a biotinylated derivative isothiocyanate with an excess (100 μM) of sulforaphane. Immunoblots showed loss of the biotin label in the macrophage inhibitor factor (MIF) by pre-incubation with sulforaphane in Nalm-6 (B-ALL) and KOPTK1 (T-ALL) cell lines. Although MIF has been targeted in colon cancer, gallbladder cancer, thyroid and squamous carcinomas, the anti-leukemic properties of MIF inhibition in blood cancers have not been explored yet. Prior to testing the effect of MIF inhibition with small molecules, we confirmed that MIF and its receptor CD74 are expressed in a panel of lymphoid (Nalm-6, REH, RS4, Jurkat, DND41, KOPTK-1) and myeloid (K562, HL60, U937, Kasumi, HEL) cell lines. The selective MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) induced cell death in a dose-dependent manner in myeloid and lymphoid cell lines but not significantly in LCL lymphoblastoid cells used as non-leukemic control; acute lymphoblastic leukemia cell lines were more sensitive to 4-IPP inhibition than myeloid leukemia cell lines. Annexin V staining showed increased apoptosis upon treatment with 4-IPP and analysis of DNA content by propidium iodide staining revealed a dose dependent arrest in S-phase. In contrast to sulforaphane and consistent with a cell cycle deregulation, immunoblot analysis showed increased cyclin E1 and a reduction of Cyclin B1. Finally, the capacity of 4-IPP to control leukemia burden in vivo will be evaluated in a cell-based xenograft model. Altogether, MIF emerges as a novel therapeutic target in leukemia that can be targeted with small molecule inhibitors.