Lunasin is a novel peptide originally identified in soybean that suppresses chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. Since the lunasin gene was cloned from soybean and the chemically synthesized form of the lunasin peptide has been used in experiments conducted so far, the isolation of lunasin from other natural sources and testing of its biological properties have not been carried out. We report here the isolation, purification, and biological assay of lunasin from barley, a newly found rich source of the peptide. The identity of lunasin was established by Western blot analysis and mass spectrometric peptide mapping of the in-gel tryptic digest of the putative protein band. Lunasin was partially purified with anion exchange and immunoaffinity chromatography. The crude and partially purified lunasin from barley suppressed colony formation in stably ras-transfected mouse fibroblast cells induced with IPTG. These fractions also inhibited histone acetylation in mouse fibroblast NIH 3T3 and human breast MCF-7 cells in the presence of the histone deacetylase inhibitor sodium butyrate.

Lunasin, a novel and promising chemopreventive compound isolated from soybean cotyledon, is a 43-amino acid peptide that contains a -RGD-cell adhesion motif followed by 8 aspartic acid residues at the carboxyl end and a structurally conserved helix region. We showed previously that lunasin peptide applied exogenously reduces foci formation in mouse fibroblast cells treated with chemical carcinogens and inhibits skin tumorigenesis induced by chemical carcinogens in mice when applied topically. In this study, lunasin peptide applied to cell culture suppresses foci formation in E1A-transfected mouse fibroblast NIH 3T3 cells. Within 18 h of exogenous application, lunasin internalizes into the cell and localizes in the nucleus. In an initial study of genes affected by lunasin, the peptide increases p21 protein levels fivefold in cells transfected with E1A but not in untransfected cells. In contrast to its inhibitory effects on cell transformation, lunasin has no effect on growth of imicroMortalized (nontumorigenc) and established cancer cells. This is the first report that lunasin suppresses transformation of mamicroMalian cells induced by an oncogene (E1A) in addition to chemical carcinogens.

Lunasin is a novel and promising chemopreventive peptide from soybean. We have shown previously that lunasin suppresses transformation of mammalian cells caused by chemical carcinogens and inhibits skin carcinogenesis in mice when applied topically. Although the lunasin gene was cloned from soybean, all experiments carried out so far in our lab have used synthetic lunasin and therefore there is no detailed description of natural lunasin isolated from soybean. We report here the first characterization of soybean lunasin that includes definitive identification by mass peptide mapping, partial purification, and measurement of bioactivities of the various purified fractions and protein expression in the developing seed. The identity of lunasin in the seed extracts was established by Western blot analysis and mass spectrometric peptide mapping. All lunasin fractions partially purified by anion exchange and immunoaffinity column chromatography suppress colony formation induced by the ras-oncogene and inhibit core H3-histone acetylation. During seed development, lunasin peptide appears 5 weeks after flowering and persists in the mature seed. Western blot analysis of different soybean varieties and commercially available soy proteins shows the presence of the peptide in varying amounts. These results demonstrate the feasibility of producing large quantities of natural lunasin from soybean for animal and human studies.

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

Despite the remarkable clinical responses achieved with BCR-ABL tyrosine kinase inhibitors (TKIs) in the treatment of chronic phase-chronic myeloid leukemia (CML), these TKIs have been less effective as single agents in blast phase (BP) CML. Identification of new therapeutic strategies is needed for the better clinical management of BP-CML. It is well known that the mitochondrial metabolic properties of tumor cells are different from those of normal cells, making this as an attractive target for cancer treatment. Previously, we screened a number of antimicrobial drugs with possible mechanisms of action related to mitochondrial metabolism and identified mefloquine as a potential candidate for CML treatment. Mefloquine is a FDA-approved antimalarial drug and has been reported to have anti-cancer activities. In this work, we investigated the effect of mefloquine and its underlying mechanisms in CML. We show that mefloquine induces apoptosis of CML cells in a dose-dependent manner (Fig. 1A). In addition, mefloquine is also effective in targeting BP-CML CD34+ progenitor cells. It induces apoptosis, inhibits colony formation and self-renewal capacity of CD34+ cells derived from a TKI-resistant BP-CML patient (Fig. 2). Mefloquine significantly enhanced anti-proliferative and pro-apoptotic effects of imatinib and dasatinib in CML cell lines as well as BP-CML CD34 cells, suggesting that mefloquine augments the effects of BCR-ABL TKIs (Fig. 1B, 2A and 2B). Mechanistically, we show that mefloquine significantly induces oxidative stress by increasing levels of mitochondrial superoxidase in K562 cells (Fig, 1C). Consistent with this, mefloquine disrupts lysosomal integrity/function in CML cells as measured by LysoTracker labelling (Fig. 1D). Taken together, we demonstrate that mefloquine is active against BP-CML and enhances the efficacy of BCR-ABL TKIs. Our work also highlights the therapeutic value of targeting oxidative stress and lysosome in the treatment of BP-CML. Figure 1 Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 1. Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 2 Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Figure 2. Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Disclosures Hwang: Sanofi: Honoraria, Other: Travel support; Janssen: Honoraria, Other: Travel support; BMS: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Pfizer: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support. Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Chiltern: Honoraria.

Acute myeloid leukemia (AML) is a complex hematological malignancy characterized by extensive heterogeneity in genetics, response to therapy and long-term outcomes, making it a prototype example of development for personalized medicine. Given the accessibility to hematologic malignancy patient samples and recent advances in high-throughput technologies, large amounts of biological data that are clinically relevant for diagnosis, risk stratification and targeted drug development have been generated. Recent studies highlight the potential of implementing genomic-based and phenotypic-based screens in clinics to improve survival in patients with refractory AML. In this review, we will discuss successful applications as well as challenges of most up-to-date high-throughput technologies, including artificial intelligence (AI) approaches, in the development of personalized medicine for AML, and recent clinical studies for evaluating the utility of integrating genomics-guided and drug sensitivity testing-guided treatment approaches for AML patients.

The resistance of chronic myeloid leukaemia (CML) to tyrosine kinase inhibitors (TKIs) remains a significant clinical problem. Targeting alternative pathways, such as protein prenylation, is known to be effective in overcoming resistance. Simvastatin inhibits 3-hydroxy-3-methylglutaryl-CoA reductase (a key enzyme in isoprenoid-regulation), thereby inhibiting prenylation. We demonstrate that simvastatin alone effectively inhibits proliferation in a panel of TKI-resistant CML cell lines, regardless of mechanism of resistance. We further show that the combination of nilotinib and simvastatin synergistically kills CML cells via an increase in apoptosis and decrease in prosurvival proteins and cellular proliferation. Mechanistically, simvastatin inhibits protein prenylation as shown by increased levels of unprenylated Ras and rescue experiments with mevalonate resulted in abrogation of synergism. The combination also leads to an increase in the intracellular uptake and retention of radiolabelled nilotinib, which further enhances the inhibition of Bcr-Abl kinase activity. In primary CML samples, this combination inhibits clonogenicity in both imatinib-naive and resistant cells. Such combinatorial effects provide the basis for utilising these Food and Drug Administrationapproved drugs as a potential clinical approach in overcoming resistance and improving CML treatment.

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