Inhibition of Oxidized Nucleotide Sanitation By TH1579 and Conventional Chemotherapy Cooperatively Enhance Oxidative DNA Damage and Survival in AML.

Currently, the majority of patients with acute myeloid leukemia (AML) still die of their disease due to primary resistance or relapse toward conventional reactive oxygen species (ROS)- and DNA damage-inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of patients with AML and the inv(16)/KITD816Y AML mouse model mimicking the genetics of patients with AML exhibiting poor response to standard chemotherapy (i.e., anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared with untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in patients with AML. Moreover, other cancer entities treated with ROS- and DNA damage-inducing chemo- or radiotherapies might benefit therapeutically from complementary treatment with TH1579.

Presence of Ceramidase Activity in Electronegative LDL.

Electronegative low-density lipoprotein (LDL(-)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(-). However, these enzymes' activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(-) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(-). In addition, LDL(-) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity's association with LDL(-). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(-).

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Targeting of PI3K/AKT signaling and DNA damage response in acute myeloid leukemia: a novel therapeutic strategy to boost chemotherapy response and overcome resistance.

Resistance of cancer patients to DNA damaging radiation therapy and chemotherapy remains a major problem in the clinic. The current review discusses the molecular mechanisms of therapy resistance in acute myeloid leukemia (AML) conferred by cooperative chemotherapy-induced DNA damage response (DDR) and mutational activation of PI3K/AKT signaling. In addition, strategies to overcome resistance are discussed, with particular focus on studies underpinning the vast potential of therapies combining standard chemotherapy AML regimens with small molecule inhibitors targeting key regulatory hubs at the interface of DDR and oncogenic signaling pathways.

Targeted inhibition of cooperative mutation- and therapy-induced AKT activation in AML effectively enhances response to chemotherapy.

Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.

The Role of Distinctive Sphingolipids in the Inflammatory and Apoptotic Effects of Electronegative LDL on Monocytes.

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(-)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(-) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP), and N,N-dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(-) had an increased content in Cer and Sph compared to LDL(+). LDL(-)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(-) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(-). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(-) on monocytes, whereas Sph plays a more important role in LDL(-)-induced inflammation, and S1P counteracts apoptosis.

Increased inflammatory effect of electronegative LDL and decreased protection by HDL in type 2 diabetic patients.

BACKGROUND AND AIMS: Type 2 diabetic patients have an increased proportion of electronegative low-density lipoprotein (LDL(-)), an inflammatory LDL subfraction present in blood, and dysfunctional high-density lipoprotein (HDL). We aimed at examining the inflammatory effect of LDL(-) on monocytes and the counteracting effect of HDL in the context of type 2 diabetes. METHODS: This was a cross-sectional study in which the population comprised 3 groups (n = 12 in each group): type 2 diabetic patients with good glycaemic control (GC-T2DM patients), type 2 diabetic patients with poor glycaemic control (PC-T2DM), and a control group. Total LDL, HDL, and monocytes were isolated from plasma of these subjects. LDL(-) was isolated from total LDL by anion-exchange chromatography. LDL(-) from the three groups of subjects was added to monocytes in the presence or absence of HDL, and cytokines released by monocytes were quantified by ELISA. RESULTS: LDL(-) proportion and plasma inflammatory markers were increased in PC-T2DM patients. LDL(-) from PC-T2DM patients induced the highest IL1ß, IL6, and IL10 release in monocytes compared to LDL(-) from GC-T2DM and healthy subjects, and presented the highest content of non-esterified fatty acids (NEFA). In turn, HDL from PC-T2DM patients showed the lowest ability to inhibit LDL(-)-induced cytokine release in parallel to an impaired ability to decrease NEFA content in LDL(-). CONCLUSIONS: Our findings show an imbalance in the pro- and anti-inflammatory effects of lipoproteins from T2DM patients, particularly in PC-T2DM.

Inflammatory intracellular pathways activated by electronegative LDL in monocytes.

AIMS: Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes. METHODS AND RESULTS: We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. CONCLUSIONS: LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes.

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL / La LDL enriquecida en ceramida induce la liberación de citoquinas a través de TLR4 y CD14 en monocitos. Similitudes con la LDL electronegativa

Introduction: In vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(-)), a heterogeneous subfraction of LDL found in plasma. LDL(-) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. Aim To evaluate TLR4 andCD14 as the putative receptors involved in cytokine release induced by CER-LDL. Methods CER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(-) were incubated for 20 h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. Results: The release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. Conclusion: CER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(-). The increased content of CER in LDL(-) is then related to the inflammatory action of LDL(-)

Introducción: La LDL enriquecida in vitro en ceramida (CER-LDL) reproduce varias características atribuidas a la LDL electronegativa (LDL(-)), subfracción heterogénea de LDL presente en circulación que induce la liberación de citoquinas en monocitos mediante CD14 y TLR4. La CER-LDL estimula también la liberación de citoquinas en monocitos, aunque el mecanismo se desconoce. Objetivo: Evaluar si CD14-TLR4 son receptores activados por la CER-LDL para inducir la liberación de citoquinas. Material y métodos: La CER-LDL se obtuvo in vitro mediante incubación de LDL nativa con liposomas enriquecidos en CER. El contenido en CER de la CER-LDL fue evaluado mediante cromatografía en capa fina. La CER-LDL y la LDL(−) fueron incubadas 20 h con monocitos humanos en presencia o ausencia de un inhibidor de la señalización de TLR4. También se incubaron con 2 líneas de monocitos humanos, THP1 y THP1, que sobreexpresan CD14 (THP1-CD14). Se evaluaron IL-6, IL-10 y MCP-1 en todos los sobrenadantes celulares mediante ELISA. Resultados: La liberación de IL-6, IL-10 y MCP-1 inducida por la CER-LDL en monocitos fue inhibida mediante VIPER (90% de inhibición), inhibidor específico de TLR4. Las citoquinas liberadas por la CER-LDL fueron escasas en THP1, células que presentan baja expresión de CD14. Las citoquinas liberadas por la CER-LDL en THP1-CD14 fueron superiores y dependientes del contenido en CER de la LDL. Conclusión: La LDL-CER induce IL-6, IL-10 y MCP-1 a través de la activación de CD14-TLR4 en monocitos, mimetizando a la LDL(-). La acción inflamatoria de la LDL(-) está relacionada con su contenido aumentado en CER

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL.

INTRODUCTION: In vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(-)), a heterogeneous subfraction of LDL found in plasma. LDL(-) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. AIM: To evaluate TLR4 and CD14 as the putative receptors involved in cytokine release induced by CER-LDL. METHODS: CER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(-) were incubated for 20 h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. RESULTS: The release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by CER-LDL was negligible in THP1, cells presenting a very low CD14 expression. In contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. CONCLUSION: CER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(-). The increased content of CER in LDL(-) is then related to the inflammatory action of LDL(-).

Electronegative LDL: a circulating modified LDL with a role in inflammation.

Electronegative low density lipoprotein (LDL(-)) is a minor modified fraction of LDL found in blood. It comprises a heterogeneous population of LDL particles modified by various mechanisms sharing as a common feature increased electronegativity. Modification by oxidation is one of these mechanisms. LDL(-) has inflammatory properties similar to those of oxidized LDL (oxLDL), such as inflammatory cytokine release in leukocytes and endothelial cells. However, in contrast with oxLDL, LDL(-) also has some anti-inflammatory effects on cultured cells. The inflammatory and anti-inflammatory properties ascribed to LDL(-) suggest that it could have a dual biological effect.

Eplerenone in systemic right ventricle: double blind randomized clinical trial. The evedes study.

BACKGROUND: There is no proven pharmacological strategy for the treatment of the failing systemic right ventricle (SRV) but myocardial fibrosis may play a role in its pathophysiology. METHODS: We designed a double-blind, placebo-controlled clinical trial to assess the effects of eplerenone 50mg during 12 months on cardiac magnetic resonance parameters (SRV mass and ejection fraction) and neurohormonal and collagen turnover biomarker (CTB) levels. RESULTS: Twenty six patients with atrial switch repair for transposition of the great arteries were randomized to eplerenone (n=14) or placebo (n=12) and 14 healthy volunteers served as controls for comparison of baseline neurohormones and CTB levels. The study population showed a good baseline profile in terms of SRV mass (57.4 ± 17 g/m(2)) and ejection fraction (54.9 ± 7.5%). However, levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), C terminal propeptide of type I procollagen (CICP) and C-terminal Telopeptide of type I Collagen (ICTP) were significantly elevated when compared to healthy controls. After one year of treatment, a trend toward reduction of CICP, N-terminal pro-Matrix Metalloproteinase 1 (NT-proMMP1), Tissue Inhibitor of Metalloproteinases 1 (TIMP1) and galectin 3 levels and a lower increase in ICTP in patients under eplerenone was observed. The reduction of SRV mass and the improvement of SRV function with eplerenone were not conclusive. CONCLUSIONS: Patients with SRV treated with eplerenone showed an improvement of an altered baseline CTB profile suggesting that reduction of myocardial fibrosis might be a therapeutic target in these patients.

CD14 and TLR4 mediate cytokine release promoted by electronegative LDL in monocytes.

AIMS: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes. METHODS AND RESULTS: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells. CONCLUSIONS: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations.

The Induction of Cytokine Release in Monocytes by Electronegative Low-Density Lipoprotein (LDL) Is Related to Its Higher Ceramide Content than Native LDL.

Electronegative low-density lipoprotein (LDL(-)) is a minor modified LDL subfraction that is present in blood. LDL(-) promotes inflammation and is associated with the development of atherosclerosis. We previously reported that the increase of cytokine release promoted by this lipoprotein subfraction in monocytes is counteracted by high-density lipoprotein (HDL). HDL also inhibits a phospholipase C-like activity (PLC-like) intrinsic to LDL(-). The aim of this work was to assess whether the inhibition of the PLC-like activity by HDL could decrease the content of ceramide (CER) and diacylglycerol (DAG) generated in LDL(-). This knowledge would allow us to establish a relationship between these compounds and the inflammatory activity of LDL(-). LDL(-) incubated at 37 °C for 20 h increased its PLC-like activity and, subsequently, the amount of CER and DAG. We found that incubating LDL(-) with HDL decreased both products in LDL(-). Native LDL was modified by lipolysis with PLC or by incubation with CER-enriched or DAG-enriched liposomes. The increase of CER in native LDL significantly increased cytokine release, whereas the enrichment in DAG did not show these inflammatory properties. These data point to CER, a resultant product of the PLC-like activity, as a major determinant of the inflammatory activity induced by LDL(-) in monocytes.