Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes.

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.

Sphingolipids as Regulators of Neuro-Inflammation and NADPH Oxidase 2.

Neuro-inflammation accompanies numerous neurological disorders and conditions where it can be associated with a progressive neurodegenerative pathology. In a similar manner, alterations in sphingolipid metabolism often accompany or are causative features in degenerative neurological conditions. These include dementias, motor disorders, autoimmune conditions, inherited metabolic disorders, viral infection, traumatic brain and spinal cord injury, psychiatric conditions, and more. Sphingolipids are major regulators of cellular fate and function in addition to being important structural components of membranes. Their metabolism and signaling pathways can also be regulated by inflammatory mediators. Therefore, as certain sphingolipids exert distinct and opposing cellular roles, alterations in their metabolism can have major consequences. Recently, regulation of bioactive sphingolipids by neuro-inflammatory mediators has been shown to activate a neuronal NADPH oxidase 2 (NOX2) that can provoke damaging oxidation. Therefore, the sphingolipid-regulated neuronal NOX2 serves as a mechanistic link between neuro-inflammation and neurodegeneration. Moreover, therapeutics directed at sphingolipid metabolism or the sphingolipid-regulated NOX2 have the potential to alleviate neurodegeneration arising out of neuro-inflammation.

Genome mining of biosynthetic and chemotherapeutic gene clusters in Streptomyces bacteria.

Streptomyces bacteria are known for their prolific production of secondary metabolites, many of which have been widely used in human medicine, agriculture and animal health. To guide the effective prioritization of specific biosynthetic gene clusters (BGCs) for drug development and targeting the most prolific producer strains, knowledge about phylogenetic relationships of Streptomyces species, genome-wide diversity and distribution patterns of BGCs is critical. We used genomic and phylogenetic methods to elucidate the diversity of major classes of BGCs in 1,110 publicly available Streptomyces genomes. Genome mining of Streptomyces reveals high diversity of BGCs and variable distribution patterns in the Streptomyces phylogeny, even among very closely related strains. The most common BGCs are non-ribosomal peptide synthetases, type 1 polyketide synthases, terpenes, and lantipeptides. We also found that numerous Streptomyces species harbor BGCs known to encode antitumor compounds. We observed that strains that are considered the same species can vary tremendously in the BGCs they carry, suggesting that strain-level genome sequencing can uncover high levels of BGC diversity and potentially useful derivatives of any one compound. These findings suggest that a strain-level strategy for exploring secondary metabolites for clinical use provides an alternative or complementary approach to discovering novel pharmaceutical compounds from microbes.

The Role of the Microbiome in Cancer and the Development of Cancer Therapeutics.

Cancer is caused by a compilation of hereditary and environmental factors. In the past decade, next-generation sequencing has revealed the extent to which the microbiome influences the maintenance of homeostasis and therefore the prevention of diseases such as cancer. Current research efforts explore the interaction between cancer and the microbiome, and the results are anticipated to transform how clinicians approach cancer treatment. There is a plausible transition from the use of human genetic biomarkers to microbiomic biomarkers for genomic diagnostics. Considering the expanding knowledge of the ways in which the microbiome can affect the development of cancer, clinicians treating cancer patients should be considerate of how the microbiome can influence the host-drug or microbiome-cancer interactions. Recognition of the importance of the microbiome within the field of oncology is pertinent to understanding and furthering cancer development and treatment.

Therapeutic effect of Northern Labrador tea extracts for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy that is one of the more common pediatric malignancies in addition to occurring with high incidence in the aging population. Unfortunately, these patient groups are quite sensitive to toxicity from chemotherapy. Northern Labrador tea, or Rhododendron tomentosum Harmaja (a.k.a. Ledum palustre subsp. decumbens) or "tundra tea," is a noteworthy medicinal plant used by indigenous peoples in Alaska, Canada, and Greenland to treat a diversity of ailments. However, laboratory investigations of Northern Labrador tea, and other Labrador tea family members, as botanical sources for anticancer compounds have been limited. Utilizing an AML cell line in both in vitro and in vivo studies, as well as in vitro studies using primary human AML patient samples, this study demonstrated for the first time that Northern Labrador tea extracts can exert anti-AML activity and that this may be attributed to ursolic acid as a constituent component. Therefore, this medicinal herb holds the potential to serve as a source for further drug discovery efforts to isolate novel anti-AML compounds.

Therapeutic Effect of Blueberry Extracts for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with high incidence in the aging population. In addition, AML is one of the more common pediatric malignancies. Unfortunately, both of these patient groups are quite sensitive to chemotherapy toxicities. Investigation of blueberries specifically as an anti-AML agent has been limited, despite being a prominent natural product with no reported toxicity. In this study, blueberry extracts are reported for the first time to exert a dietary therapeutic effect in animal models of AML. Furthermore, in vitro studies revealed that blueberry extracts exerted anti-AML efficacy against myeloid leukemia cell lines as well as against primary AML, and specifically provoked Erk and Akt regulation within the leukemia stem cell subpopulation. This study provides evidence that blueberries may be unique sources for anti-AML biopharmaceutical compound discovery, further warranting fractionation of this natural product. More so, blueberries themselves may provide an intriguing dietary option to enhance the anti-AML efficacy of traditional therapy for subsets of patients that otherwise may not tolerate rigorous combinations of therapeutics.

Epigenetics and Sphingolipid Metabolism in Health and Disease.

Sphingolipids represent one of the major classes of bioactive lipids. Studies of sphingolipids have intensified in the past several years, revealing their roles in nearly all cell biological processes. In addition, epigenetic regulation has gained substantial interest due to its role in controlling gene expression and activity without changing the genetic code. In this review, we first introduce a brief background on sphingolipid biology, highlighting its role in pathophysiology. We then illustrate the concept of epigenetic regulation, focusing on how it affects the metabolism of sphingolipids. We further discuss the roles of bioactive sphingolipids as epigenetic regulators themselves. Overall, a better understanding of the relationship between epigenetics and sphingolipid metabolism may help to improve the development of sphingolipid-targeted therapeutics.

Combinatorial Efficacy of Quercitin and Nanoliposomal Ceramide for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with limited treatment options. Inflammation is often a contributing factor to the development and progression of AML, and related diseases, and can potentiate therapy failure. Previously, we had identified anti-inflammatory roles and anti-AML efficacy for blueberry extracts. The present study extended these observations to determine that the polyphenol quercetin inhibited neutral sphingomyelinase (N-SMase) activity and exerted anti-AML efficacy. Moreover, quercetin was shown to exert combinatorial anti-AML efficacy with nanoliposomal ceramide. Overall, this demonstrated that quercetin could block the pro-inflammatory actions of N-SMase and augment the efficacy of anti-AML therapeutics, including ceramide-based therapeutics.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target.

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia.

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia--Impact on enzyme activity and response to cytotoxics.

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 µM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

Extracts of Devil's club (Oplopanax horridus) exert therapeutic efficacy in experimental models of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a group of hematological malignancies defined by expanded clonal populations of immature progenitors (blasts) of myeloid phenotype in blood and bone marrow. Given a typical poor prognostic outlook, there is great need for novel agents with anti-AML activity. Devil's club (Oplopanax horridus) is one of the most significant medicinal plants used among the indigenous people of Southeast Alaska and the coastal Pacific Northwest, with different linguistic groups utilizing various parts of the plant to treat many different conditions including cancer. Studies identifying medically relevant components in Devil's club are limited. For this research study, samples were extracted in 70% ethanol before in vitro analysis, to assess effects on AML cell line viability as well as to study regulation of tyrosine phosphorylation and cysteine oxidation. The root extract displayed better in vitro anti-AML efficacy in addition to a noted anti-tyrosine kinase activity independent of an antioxidant effect. In vivo therapeutic studies using an immunocompetent murine model of AML further demonstrated that Devil's club root extract improved the murine survival while decreasing immunosuppressive regulatory T cells and improving CD8+ T-cell functionality. This study defines for the first time an anti-AML efficacy for extracts of Devil's club.

Maritoclax induces apoptosis in acute myeloid leukemia cells with elevated Mcl-1 expression.

Acute myeloid leukemia (AML) is one of the deadliest leukemias for which there is an urgent and unmet need for the development of novel treatment strategies. Multiple drug resistance mechanisms mediate poor drug response and relapse in patients, and a selective Mcl-1 inhibitor has been speculated to be a promising agent in the treatment of AML. Here, we describe that maritoclax, a small molecule Mcl-1 inhibitor, induces Mcl-1 proteasomal degradation without transcriptional downregulation. Maritoclax killed AML cell lines and primary cells with elevated Mcl-1 levels through selective Mcl-1 downregulation, and synergized with ABT-737 to overcome Mcl-1-mediated ABT-737 resistance. Maritoclax was more effective than daunorubicin at inducing leukemic cell death when co-cultured with HS-5 bone marrow stroma cells, while being less toxic than daunorubicin against HS-5 stroma cells, primary mouse bone marrow cells, and hematopoietic progenitor cells. Moreover, maritoclax administration at 20 mg/kg/d intraperitoneally caused significant U937 tumor shrinkage, as well as 36% tumors remission rate in athymic nude mice, without apparent toxicity to healthy tissue or circulating blood cells. In summary, our studies suggest that maritoclax belongs to a novel class of Mcl-1 inhibitors that has the potential to be developed for the treatment of AML.

Cholecystokinin mediates progression and metastasis of pancreatic cancer associated with dietary fat.

BACKGROUND: Obesity and dietary fat are associated with increased risk of several malignancies including pancreatic cancer. The incidence of pancreatic cancer is increased in countries that consume diets high in fat. AIM: The purpose of this study was to assess the relationship and mechanism of action between dietary fat and endogenous cholecystokinin (CCK) on pancreatic tumor growth and metastasis in an immunocompetent animal model. METHODS: C57BL/6 mice were placed on regular, low-fat, or high-fat diets for 8 weeks before establishment of Panc-02 orthotopic pancreatic tumors. Mice were then treated with a CCK-A receptor antagonist, devazepide, or vehicle for an additional 2.5 weeks. Pancreas tumors were weighed and metastases counted. Blood CCK levels were measured by radioimmunoassay (RIA). Tissues were examined histologically and studied for genes associated with metastasis by RT-PCR array. Effects of the CCK antagonist on Panc-02 cells invasiveness was assessed in a Matrigel invasion assay. RESULTS: Mice that received the high-fat diet had larger tumors and tenfold higher serum CCK levels by RIA compared to normal diet controls (p < 0.01). Pancreatic tumors in high-fat diet mice treated with the antagonist had fewer intravascular tumor emboli and metastases compared to controls. The reduction in tumor emboli correlated with decreased vascular endothelial growth factor-A (VEGF-A) expression in tumors (p < 6 × 10(-9)). In vitro invasiveness of Panc-02 cells also was reduced by CCK-A receptor antagonist treatment (p = 1.33 × 10(-6)). CONCLUSION: CCK is a mediator of dietary fat-associated pancreatic cancer. CCK is also involved in the invasiveness of pancreatic tumors through a mechanism involving VEGF-A.

Engraftment of Human Primary Acute Myeloid Leukemia Defined by Integrated Genetic Profiling in NOD/SCID/IL2rγnull Mice for Preclinical Ceramide-Based Therapeutic Evaluation.

Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few available therapeutic options. Novel advances are urgently needed, however effective models to test experimental therapeutics have been lacking. Recently, NOD/SCID/IL2rγnull (NSG) mice were shown to engraft primary human AML in a manner that recapitulated the natural disease and its progression. Additionally, integrated genomic profiling was used to refine risk stratification of AML. In this study, we demonstrated the engraftment of molecularly defined primary AML in NSG mice. We showed that AML that express DNMT3A mutations, which predict for adverse outcome, engrafted with exceptional efficacy. Lastly, we demonstrated that human AML-engrafted NSG mice can be effectively used to study novel ceramide-based therapeutics. Ceramide is a bioactive sphingolipid that has been implicated as an inducer of apoptosis. Elevation in cancer cell ceramide levels either via exogenous delivery or by provoking intracellular ceramide generation is the goal of ceramide-based therapeutics. In this study, we used the human AML-engrafted NSG mouse model to evaluate nanoliposomal short-chain C6-ceramide and a nanoliposomal formulation of the ceramide-inducer tamoxifen. Altogether, the NSG model is likely to prove invaluable in the study of novel agents, sushc as ceramide-based therapeutics, with the ability to define therapeutic activity against specific molecularly defined and risk stratified AML.

Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2-p11 heterotetrameric protein complex.

The bioactive sphingolipid, ceramide 1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid-binding ELISA, and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. Although the annexin a2-p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine 1-phosphate, and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2-p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.

The therapeutic potential of nanoscale sphingolipid technologies.

Nanotechnologies, while small in size, widen the scope of drug delivery options for compounds with problematic pharmacokinetics, such as bioactive sphingolipids. We describe the development of historical sphingolipid nanotechnologies, such as nanoliposomes, and project future uses for a broad repertoire of nanoscale sphingolipid therapy formulations. In particular, we describe sphingo-nanotherapies for treatment of cancer, inflammatory disease, and cardiovascular disease. We conclude with a discussion of the challenges associated with regulatory approval, scale-up, and development of these nanotechnology therapies for clinical applications.