FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

Regulation of cancer cell glucose metabolism is determinant for cancer cell fate after melatonin administration.

Several oncogenic pathways plus local microenvironmental conditions, such as hypoxia, converge on the regulation of cancer cells metabolism. The major metabolic alteration consists of a shift from oxidative phosphorylation as the major glucose consumer to aerobic glycolysis, although most of cancer cells utilize both pathways to a greater or lesser extent. Aerobic glycolysis, together with the directly related metabolic pathways such as the tricarboxylic acid cycle, the pentose phosphate pathway, or gluconeogenesis are currently considered as therapeutic targets in cancer research. Melatonin has been reported to present numerous antitumor effects, which result in a reduced cell growth. This is achieved with both low and high concentrations with no relevant side effects. Indeed, high concentrations of this indolamine reduce proliferation of cancer types resistant to low concentrations and induce cell death in some types of tumors. Previous work suggest that regulation of glucose metabolism and other related pathways play an important role in the antitumoral effects of high concentration of melatonin. In the present review, we analyze recent work on the regulation by such concentrations of this indolamine on aerobic glycolysis, gluconeogenesis, the tricarboxylic acid cycle and the pentose phosphate pathways of cancer cells.

Role of glucose metabolism in the differential antileukemic effect of melatonin on wild­type and FLT3­ITD mutant cells.

The FMS­like tyrosine kinase 3 internal tandem duplication (FLT3­ITD) mutation represents the most frequent genetic alteration in acute myeloid leukemia (AML) and is associated with poor prognosis. The mutation promotes cancer cell survival and proliferation, and shifts their glucose metabolism towards aerobic glycolysis, a frequent alteration in cancer. In the present study, the impact of melatonin on the viability of AML cell lines with (MV­4­11 and MOLM­13) or without the FLT3­ITD mutation (OCI­AML3 and U­937) was evaluated. Melatonin induces cell death in AML cells carrying the FLT3­ITD mutation, but only inhibits the proliferation of AML cells without this mutation. Consistently, melatonin decreases tumor growth and increases animal survival in a xenograft model of FLT3­ITD AML. Toxicity is related to a decrease in glucose uptake, lactate dehydrogenase activity, lactate production and hypoxia­inducible factor­1α activation. Melatonin also regulates the expression of glucose metabolism­related genes, impairing the balance between anaplerosis and cataplerosis, through the upregulation of the expression of phosphoenolpyruvate carboxykinase 2 (PCK2). Collectively, the present findings highlight the regulation of glucose metabolism, currently considered a possible therapeutic target in cancer, as a key event in melatonin­induced cytotoxicity, suggesting its potential as a therapeutic tool for the treatment of patients with AML, particularly those carrying the FLT3­ITD mutation that results in low basal expression levels of PCK2.

Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts Potent Activity in Preclinical Models of Acute Myeloid Leukemia.

Internal tandem duplication (ITD) or tyrosine kinase domain mutations of FLT3 is the most frequent genetic alteration in acute myelogenous leukemia (AML) and are associated with poor disease outcome. Despite considerable efforts to develop single-target FLT3 drugs, so far, the most promising clinical response has been achieved using the multikinase inhibitor midostaurin. Here, we explore the activity of the indolocarbazole EC-70124, from the same chemical space as midostaurin, in preclinical models of AML, focusing on those bearing FLT3-ITD mutations. EC-70124 potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases. EC-70124 inhibits proliferation of AML cell lines, inducing cell-cycle arrest and apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding compared with midostaurin. Both in vitro and in vivo pharmacodynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy, reaching complete tumor regression. Ex vivo, EC-70124 impaired cell viability in leukemic blasts, especially from FLT3-ITD patients. Our results demonstrate the ability of EC-70124 to reduce proliferation and induce cell death in AML cell lines, patient-derived leukemic blast and xenograft animal models, reaching best results in FLT3 mutants that carry other molecular pathways' alterations. Thus, its unique inhibition profile warrants EC-70124 as a promising agent for AML treatment based on its ability to interfere the complex oncogenic events activated in AML at several levels. Mol Cancer Ther; 17(3); 614-24. ©2018 AACR.

Distinct roles of N-acetyl and 5-methoxy groups in the antiproliferative and neuroprotective effects of melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine.

Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells.

Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.

Involvement of autophagy in melatonin-induced cytotoxicity in glioma-initiating cells.

Glioblastoma-initiating cells (GICs) represent a stem cell-like subpopulation within malignant glioblastomas responsible for tumor development, progression, therapeutic resistance, and tumor relapse. Thus, eradication of this subpopulation is essential to achieve stable, long-lasting remission. We have previously reported that melatonin decreases cell proliferation of glioblastoma cells both in vitro and in vivo and synergistically increases effectiveness of drugs in glioblastoma cells and also in GICs. In this study, we evaluated the effect of the indolamine alone in GICs and found that melatonin treatment reduces GICs proliferation and induces a decrease in self-renewal and clonogenic ability accompanied by a reduction in the expression of stem cell markers. Moreover, our results also indicate that melatonin treatment, by modulating stem cell properties, induces cell death with ultrastructural features of autophagy. Thus, data reported here reinforce the therapeutic potential of melatonin as a treatment of malignant glioblastoma both by inhibiting tumor bulk proliferation or killing GICs, and simultaneously enhancing the effect of chemotherapy.

Mechanisms involved in the pro-apoptotic effect of melatonin in cancer cells.

It is well established that melatonin exerts antitumoral effects in many cancer types, mostly decreasing cell proliferation at low concentrations. On the other hand, induction of apoptosis by melatonin has been described in the last few years in some particular cancer types. The cytotoxic effect occurs after its administration at high concentrations, and the molecular pathways involved have been only partially determined. Moreover, a synergistic effect has been found in several cancer types when it is administered in combination with chemotherapeutic agents. In the present review, we will summarize published work on the pro-apoptotic effect of melatonin in cancer cells and the reported mechanisms involved in such action. We will also construct a hypothesis on how different cell signaling pathways may relate each other on account for such effect.

Cooperative action of JNK and AKT/mTOR in 1-methyl-4-phenylpyridinium-induced autophagy of neuronal PC12 cells.

Parkinson's disease has been widely related to both apoptosis and oxidative stress. Many publications relate the loss of mitochondrial potential to an apoptosis-mediated cell death in different in vivo and in vitro models of this pathology. The present study used the dopaminegic specific neurotoxin 1-methyl-4-phenylpyridinium (MPP(+) ) on neuron-like PC12 cells, which is a well-accepted model of Parkinson's disease. Results showed an early increase in oxidants, which drives the modulation of c-Jun N-terminal kinase (JNK) and AKT/mammalian target of rapamycin (mTOR) pathways, mimicking peroxide treatment. However, the cell death found in neuronal PC12 cells treated with MPP(+) was not a caspase-associated apoptosis. Electron microscopic images illustrated autophagic cell death, which was confirmed by a Beclin-1 and ATG expression increase, accumulation of acidic vesicles, and rescue by an autophagy inhibitor. In conclusion, the boost in oxidants from MPP(+) treatment in neuronal PC12 is modulating both survival (AKT/mTOR) and death (JNK) pathways, which are the perpetrators of an autophagic cell death.

Intracellular redox state as determinant for melatonin antiproliferative vs cytotoxic effects in cancer cells.

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.