Autofagija i metaboličke promjene u diferencijaciji staničnih linija akutne mijeloične leukemije [Autography and metabolic changes in differentiation of acute myeloid leukemia cell lines]

Pharmacological modulators of metabolism and AMP-dependent kinase (AMPK) inhibit proliferation of tumor cells. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a compound commonly used as an AMPK-modulator, induced AMPK-independent differentiation of U937 cells. Autophagy has been described as an AMPK-independent effect of AICAR in other cells. Therefore, the aim of this study was to determine the role of autophagy and metabolism in differentiation of U937 cells. The results showed that AICAR-mediated effects were not mimicked by specific AMPK agonist and that AICAR had no significant effects on aerobic glycolysis. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II. These effects were not observed in response to metformin, an AMPK agonist without differentiative properties. The increase in LC3B-II was due to the increase in autophagy flux and the autophagy inhibitor 3-methyladenine inhibited differentiation in response to all inducers. The effects of AICAR and ATRA on differentiation markers did not depend on Beclin-1, hVps34 and Atg7. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that differentiation does not depend on the classical or canonical autophagy pathway

Metabolism and differentiation

Textbook biochemical pathways do not usually apply to intermediary metabolism of highly proliferating, differentiating, or tumor cells. Over 80 years ago, Otto Warburg observed that cancer cells, unlike normal cells, favor glycolysis for energy production, even under aerobic conditions, and proposed that this shift in cancer cell metabolism (termed „aerobic glycolysis”) was due to mitochondrial dysfunction. Recent studies by several groups suggest that aerobic glycolysis in tumor cells is actually caused by oncogene-directed changes in metabolism that are necessary for both continuous proliferation and a block in cellular differentiation. Phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is one of the principal proliferative and anti-apoptotic signaling pathways, which is known to support glycolysis and anabolism. Our previous studies demonstrated the activation of PI3K and Akt in nuclei of leukemia cells during differentiation, and confirmed that the inhibition of proximal components of the pathway inhibits proliferation, but negatively affects differentiative capacity of the cells. In contrast, use of rapamycin, which inhibits mTOR, a more distal component of the pathway, potentiates differentiation along granulocytic pathway. To further investigate the role of upstream regulators of mTOR in leukemia differentiation, we tested the effects of modulators of AMP-activated protein kinase (AMPK). Our results suggest a strong differentiative property of an AMPK activator, AICAR (5-amino-1-b-D-ribofuranosyl-imidazole-4-carboxamide) in monocytic U937 cells. The mechanism of AICAR-mediated effects will be presented and a possible role of AMPK-modulators in differentiation therapy will be discussed

The mechanism of synergistic effects of arsenic trioxide and rapamycin in acute myeloid leukemia cell lines lacking typical t(15;17) translocation

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G(0)/G(1) phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phosphorylated ERK, Ser473 phosphorylated Akt and anti-apoptotic Mcl-1 protein. Primary AML cells show high sensitivity to growth-inhibitory effects of rapamycin alone or in combination with ATO. The results of the present study reveal the mechanism of the synergistic effects of two drugs at therapeutically achievable doses in non-APL AML cells

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax

Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death

Autography and metabolic changes in differentiation of acute myeloid leukemia cell lines

Farmakološki modulatori metabolizma i AMP-ovisne kinaze (AMPK) koče proliferaciju tumorskih stanica. U prethodnom radu pokazali smo da 5-aminoimidazol-4-karboksamid ribonukleotid (AICAR), spoj koji se često rabi kao agonist AMPK-a, potiče diferencijaciju stanica U937 neovisno o AMPK-u. Autofagija je opisana kao jedan od AMPK-neovisnih učinaka AICAR-a u drugim stanicama. Stoga je cilj ovog istraživanja bio odrediti ulogu autofagije i metabolizma u diferencijaciji stanica U937. Rezultati su pokazali da specifični aktivator AMPK-a ne oponaša učinke AICAR-a te da AICAR nema značajno djelovanje na aerobnu glikolizu. Dugotrajna inkubacija stanica U937 s AICAR-om i drugim diferencirajućim tvarima, sve-trans-retinskom kiselinom (ATRA-om) i forbol 12-miristatom 13-acetatom (PMA-om), povećala je izražaj biljega autofagije LC3B-II, a takvi učinci nisu zamijećeni u stanicama koje su inkubirane s metforminom, agonistom AMPK-a koji ne potiče diferencijaciju. Povećanje LC3B-II posljedica je povećanog protoka autofagije, a inhibitor autofagije 3-metiladenin je u potpunosti zakočio diferencijaciju u odgovoru na sve korištene diferencirajuće tvari. Učinci AICAR-a i ATRA-e na izražaj diferencijacijskih biljega ne ovise o količini Beclina-1, hVps34 i Atg7. Ovi rezultati pokazuju da AICAR i druge diferencirajuće tvari potiču autofagijski protok u stanicama U937 te da diferencijacija ne ovisi o klasičnom ili kanonskom putu autofagije.Pharmacological modulators of metabolism and AMP-dependent kinase (AMPK) inhibit proliferation of tumor cells. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a compound commonly used as an AMPK-modulator, induced AMPK-independent differentiation of U937 cells. Autophagy has been described as an AMPK-independent effect of AICAR in other cells. Therefore, the aim of this study was to determine the role of autophagy and metabolism in differentiation of U937 cells. The results showed that AICAR-mediated effects were not mimicked by specific AMPK agonist and that AICAR had no significant effects on aerobic glycolysis. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II. These effects were not observed in response to metformin, an AMPK agonist without differentiative properties. The increase in LC3B-II was due to the increase in autophagy flux and the autophagy inhibitor 3-methyladenine inhibited differentiation in response to all inducers. The effects of AICAR and ATRA on differentiation markers did not depend on Beclin-1, hVps34 and Atg7. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that differentiation does not depend on the classical or canonical autophagy pathway

Autography and metabolic changes in differentiation of acute myeloid leukemia cell lines

Farmakološki modulatori metabolizma i AMP-ovisne kinaze (AMPK) koče proliferaciju tumorskih stanica. U prethodnom radu pokazali smo da 5-aminoimidazol-4-karboksamid ribonukleotid (AICAR), spoj koji se često rabi kao agonist AMPK-a, potiče diferencijaciju stanica U937 neovisno o AMPK-u. Autofagija je opisana kao jedan od AMPK-neovisnih učinaka AICAR-a u drugim stanicama. Stoga je cilj ovog istraživanja bio odrediti ulogu autofagije i metabolizma u diferencijaciji stanica U937. Rezultati su pokazali da specifični aktivator AMPK-a ne oponaša učinke AICAR-a te da AICAR nema značajno djelovanje na aerobnu glikolizu. Dugotrajna inkubacija stanica U937 s AICAR-om i drugim diferencirajućim tvarima, sve-trans-retinskom kiselinom (ATRA-om) i forbol 12-miristatom 13-acetatom (PMA-om), povećala je izražaj biljega autofagije LC3B-II, a takvi učinci nisu zamijećeni u stanicama koje su inkubirane s metforminom, agonistom AMPK-a koji ne potiče diferencijaciju. Povećanje LC3B-II posljedica je povećanog protoka autofagije, a inhibitor autofagije 3-metiladenin je u potpunosti zakočio diferencijaciju u odgovoru na sve korištene diferencirajuće tvari. Učinci AICAR-a i ATRA-e na izražaj diferencijacijskih biljega ne ovise o količini Beclina-1, hVps34 i Atg7. Ovi rezultati pokazuju da AICAR i druge diferencirajuće tvari potiču autofagijski protok u stanicama U937 te da diferencijacija ne ovisi o klasičnom ili kanonskom putu autofagije.Pharmacological modulators of metabolism and AMP-dependent kinase (AMPK) inhibit proliferation of tumor cells. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a compound commonly used as an AMPK-modulator, induced AMPK-independent differentiation of U937 cells. Autophagy has been described as an AMPK-independent effect of AICAR in other cells. Therefore, the aim of this study was to determine the role of autophagy and metabolism in differentiation of U937 cells. The results showed that AICAR-mediated effects were not mimicked by specific AMPK agonist and that AICAR had no significant effects on aerobic glycolysis. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II. These effects were not observed in response to metformin, an AMPK agonist without differentiative properties. The increase in LC3B-II was due to the increase in autophagy flux and the autophagy inhibitor 3-methyladenine inhibited differentiation in response to all inducers. The effects of AICAR and ATRA on differentiation markers did not depend on Beclin-1, hVps34 and Atg7. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that differentiation does not depend on the classical or canonical autophagy pathway

Autography and metabolic changes in differentiation of acute myeloid leukemia cell lines

Farmakološki modulatori metabolizma i AMP-ovisne kinaze (AMPK) koče proliferaciju tumorskih stanica. U prethodnom radu pokazali smo da 5-aminoimidazol-4-karboksamid ribonukleotid (AICAR), spoj koji se često rabi kao agonist AMPK-a, potiče diferencijaciju stanica U937 neovisno o AMPK-u. Autofagija je opisana kao jedan od AMPK-neovisnih učinaka AICAR-a u drugim stanicama. Stoga je cilj ovog istraživanja bio odrediti ulogu autofagije i metabolizma u diferencijaciji stanica U937. Rezultati su pokazali da specifični aktivator AMPK-a ne oponaša učinke AICAR-a te da AICAR nema značajno djelovanje na aerobnu glikolizu. Dugotrajna inkubacija stanica U937 s AICAR-om i drugim diferencirajućim tvarima, sve-trans-retinskom kiselinom (ATRA-om) i forbol 12-miristatom 13-acetatom (PMA-om), povećala je izražaj biljega autofagije LC3B-II, a takvi učinci nisu zamijećeni u stanicama koje su inkubirane s metforminom, agonistom AMPK-a koji ne potiče diferencijaciju. Povećanje LC3B-II posljedica je povećanog protoka autofagije, a inhibitor autofagije 3-metiladenin je u potpunosti zakočio diferencijaciju u odgovoru na sve korištene diferencirajuće tvari. Učinci AICAR-a i ATRA-e na izražaj diferencijacijskih biljega ne ovise o količini Beclina-1, hVps34 i Atg7. Ovi rezultati pokazuju da AICAR i druge diferencirajuće tvari potiču autofagijski protok u stanicama U937 te da diferencijacija ne ovisi o klasičnom ili kanonskom putu autofagije.Pharmacological modulators of metabolism and AMP-dependent kinase (AMPK) inhibit proliferation of tumor cells. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a compound commonly used as an AMPK-modulator, induced AMPK-independent differentiation of U937 cells. Autophagy has been described as an AMPK-independent effect of AICAR in other cells. Therefore, the aim of this study was to determine the role of autophagy and metabolism in differentiation of U937 cells. The results showed that AICAR-mediated effects were not mimicked by specific AMPK agonist and that AICAR had no significant effects on aerobic glycolysis. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II. These effects were not observed in response to metformin, an AMPK agonist without differentiative properties. The increase in LC3B-II was due to the increase in autophagy flux and the autophagy inhibitor 3-methyladenine inhibited differentiation in response to all inducers. The effects of AICAR and ATRA on differentiation markers did not depend on Beclin-1, hVps34 and Atg7. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that differentiation does not depend on the classical or canonical autophagy pathway

5-Aminoimidazole-4-carboxamide ribonucleoside-induced autophagy flux during differentiation of monocytic leukemia cells

Pharmacological modulators of AMP-dependent kinase (AMPK) have been suggested in treatment of cancer. The biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) have been reported to inhibit proliferation of solid tumors and hematological malignancies, but their role in differentiation is less explored. Our previous study demonstrated that AICAR alone induced AMPK-independent expression of differentiation markers in monocytic U937 leukemia cells, and no such effects were observed in response to metformin. The aim of this study was to determine the mechanism of AICAR-mediated effects and to test for the possible role of autophagy in differentiation of leukemia cells. The results showed that AICAR-mediated effects on the expression of differentiation markers were not mimicked by A769662, a more specific direct AMPK activator. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II, and these effects were not observed in response to metformin. Western blot and immunofluorescence analyses of U937 cells treated with bafilomycin A1 or transfected with mRFP-GFP-LC3 proved that the increase in the expression of LC3B-II was due to an increase in autophagy flux, and not to a decrease in lysosomal degradation. 3-Methyladenine inhibited the expression of differentiation markers in response to all inducers, but had stimulatory effects on autophagy flux at dose that effectively inhibited the production of phosphatidylinositol 3-phosphate. The small inhibitory RNA-mediated down-modulation of Beclin 1 and hVPS34 had no effects on AICAR and ATRA-mediated increase in the expression of differentiation markers. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that the effects of AICAR and ATRA on the expression of differentiation markers do not depend on the normal levels of key proteins of the classical or canonical autophagy pathway