Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo.

Abstract

Activating mutations of receptor tyrosine kinases (RTKs) are involved in the pathophysiology of various types of cancers. Among them, FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Although a number of clinical trials were conducted, an effective RTK inhibitor with acceptable toxicity hasn’t been developed.Recent studies have demonstrated these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules in a compartment-specific manner. So, we speculated that manipulation of their intracellular trafficking between intracellular compartments might be promising as a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of chlorpromazine (CPZ) as an inhibitor of membrane trafficking, which is a well-established antipsychotic drug. All of the protocols were approved by the Institutional Review Board of Kinki University Faculty of Medicine, and we obtained AML samples after the written informed consent was given from all the patients.First, to examine the effect of CPZ on the intracellular trafficking of RTK mutants, we introduced murine FLT3-ITD and KIT-D814V (corresponding to human KIT D816V) into murine embryonic fibroblasts (MEFs) isolated from E14.5 fetuses, respectively. Both FLT3-ITD and KIT-D814V were distributed in cytoplasm with a punctate pattern, both of which were mainly located close to endoplasmic reticulum (ER) and Golgi. In contrast, when treated with CPZ, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi in a diffused pattern, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ on cell growth and survival using several human leukemia cell lines and primary AML cells with or without mutant RTK. As a result, CPZ severely inhibited the growth and survival of leukemia cell lines and primary AML cells with mutant RTK in a dose-dependent manner (up to 95% inhibition; IC50: between 1.5 μM and 10.7 μM). In addition, CPZ inhibited the growth of the cells without mutant RTK, whereas the IC50 levels were 3- to 4-fold higher than those seen in the cells with mutant RTK. We also examined the effects of CPZ on the growth and survival of CD34+38- leukemic stem cells (LSCs) derived from AML patients (n=4), and found that CPZ effectively inhibited the growth and/or survival of LSCs, especially in LSCs with mutant RTK (IC50=7.5 μM). As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) LSCs. These results indicate that CPZ inhibits cell growth and/or survival of AML cells and LSCs harboring mutant RTKs. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. Three days after the transplantation of human AML cells with mutant RTKs (n=8), NOD/Scid/IL2Rg-KO (NOG) mice were treated with normal saline or 10 mg/kg CPZ intraperitoneally on everyday schedule. At first, we confirmed that plasma concentration levels of CPZ on this schedule were within the non-toxic range as previously reported (0.03 to 0.71 μM, n=3) and that CPZ doesn’t affect normal hematopoiesis (n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ AML cells and CD34+38- LSCs were significantly reduced by CPZ treatment in the bone marrow (BM) of the recipient mice compared with normal saline-treated control (ctrl) mice (CD45+ 58.2% vs 8.47%, p<.001; LSCs 0.51% vs 0.07%, p<.005), indicating that CPZ can inhibit the growth and survival of both AML cells and LSCs in vivo. We further tested the anti-leukemic activities of CPZ on established human AML in a zenograft model. After confirming the engraftment of human AML cells by BM sampling six weeks after transplantation of FLT3-ITD(+) AML cells (n=3), these mice were treated with CPZ or normal saline. In all cases tested, CPZ treatment for 4 weeks significantly reduced human AML fraction in the BM. (18.4% vs 1.99%, p<.005)Together, these results suggest that CPZ, an inhibitor of the intracellular trafficking of leukemogenic RTKs, would be a promising therapeutic drug to eradicate AML cells with an established safety profile. DisclosuresRai:Shionogi & Co., Ltd. : Research Funding. Tanaka:Shionogi & Co., Ltd. : Research Funding. Tanimura:Shionogi & Co., Ltd. : Research Funding. Matsui:Shionogi & Co., Ltd. : Research Funding. Kanakura:Shionogi & Co., Ltd. : Research Funding. Matsumura:Shionogi & Co., Ltd. : Research Funding.