Publications by authors named "Rachel Culp-Hill"

28 Publications

  • Page 1 of 1

Extinguishing the Embers: Targeting AML Metabolism.

Trends Mol Med 2020 Oct 26. Epub 2020 Oct 26.

Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Acute myeloid leukemia (AML) is a cancer derived from the myeloid lineage of blood cells, characterized by overproduction of leukemic blasts. Although therapeutic improvements have made a significant impact on the outcomes of patients with AML, survival rates remain low due to a high incidence of relapse. Similar to how wildfires can reignite from hidden embers not extinguished from an initial round of firefighting, leukemic stem cells (LSCs) are the embers remaining after completion of traditional chemotherapeutic treatments. LSCs exhibit a unique metabolic profile and contain metabolically distinct subpopulations. In this review, we detail the metabolic features of LSCs and how these characteristics promote resistance to traditional chemotherapy. We also discuss new therapeutic approaches that target metabolic vulnerabilities of LSC to selectively eradicate them.
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http://dx.doi.org/10.1016/j.molmed.2020.10.001DOI Listing
October 2020

Interactions between host genetics and gut microbiota determine susceptibility to CNS autoimmunity.

Proc Natl Acad Sci U S A 2020 11 19;117(44):27516-27527. Epub 2020 Oct 19.

Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT 05401;

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. The etiology of MS is multifactorial, with disease risk determined by genetics and environmental factors. An emerging risk factor for immune-mediated diseases is an imbalance in the gut microbiome. However, the identity of gut microbes associated with disease risk, their mechanisms of action, and the interactions with host genetics remain obscure. To address these questions, we utilized the principal autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), together with a genetically diverse mouse model representing 29 unique host genotypes, interrogated by microbiome sequencing and targeted microbiome manipulation. We identified specific gut bacteria and their metabolic functions associated with EAE susceptibility, implicating short-chain fatty acid metabolism as a key element conserved across multiple host genotypes. In parallel, we used a reductionist approach focused on two of the most disparate phenotypes identified in our screen. Manipulation of the gut microbiome by transplantation and cohousing demonstrated that transfer of these microbiomes into genetically identical hosts was sufficient to modulate EAE susceptibility and systemic metabolite profiles. Parallel bioinformatic approaches identified as a commensal species unexpectedly associated with exacerbation of EAE in a genetically susceptible host, which was functionally confirmed by bacterial isolation and commensal colonization studies. These results reveal complex interactions between host genetics and gut microbiota modulating susceptibility to CNS autoimmunity, providing insights into microbiome-directed strategies aimed at lowering the risk for autoimmune disease and underscoring the need to consider host genetics and baseline gut microbiome composition.
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http://dx.doi.org/10.1073/pnas.2002817117DOI Listing
November 2020

The Hepatic Microenvironment Uniquely Protects Leukemia Cells through Induction of Growth and Survival Pathways Mediated by LIPG.

Cancer Discov 2021 Feb 7;11(2):500-519. Epub 2020 Oct 7.

Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Due to the disseminated nature of leukemia, malignant cells are exposed to many different tissue microenvironments, including a variety of extramedullary sites. In the present study, we demonstrate that leukemic cells residing in the liver display unique biological properties and also contribute to systemic changes that influence physiologic responses to chemotherapy. Specifically, the liver microenvironment induces metabolic adaptations via upregulating expression of endothelial lipase in leukemia cells, which not only stimulates tumor cell proliferation through polyunsaturated fatty acid-mediated pathways, but also promotes survival by stabilizing antiapoptotic proteins. Additionally, hepatic infiltration and tissue damage caused by malignant cells induces release of liver-derived enzymes capable of degrading chemotherapy drugs, an event that further protects leukemia cells from conventional therapies. Together, these studies demonstrate a unique role for liver in modulating the pathogenesis of leukemic disease and suggest that the hepatic microenvironment may protect leukemia cells from chemotherapeutic challenge. SIGNIFICANCE: The studies presented herein demonstrate that the liver provides a microenvironment in which leukemia cells acquire unique metabolic properties. The adaptations that occur in the liver confer increased resistance to chemotherapy. Therefore, we propose that therapies designed to overcome liver-specific metabolic changes will yield improved outcomes for patients with leukemia..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858222PMC
February 2021

Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells.

Cell Stem Cell 2020 Nov 20;27(5):748-764.e4. Epub 2020 Aug 20.

Division of Hematology, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.
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http://dx.doi.org/10.1016/j.stem.2020.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655603PMC
November 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity.

Haematologica 2020 04 2. Epub 2020 Apr 2.

University of Virginia, Charlotesville, VA, USA.

Red blood cell storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBCs to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBCs from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyze following oxidant stress. A subset of extreme hemolyzers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBCs from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBCs stored in additive solution-3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBCs stored in additive solution-1. Merging metabolomics data with results from an independent GWAS study on the same cohort, we identified metabolic markers of hemolysis and G6PD-deficiency, which were associated with extremes in oxidative hemolysis and dysregulation in NADPH and glutathione-dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and G6PD status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.
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http://dx.doi.org/10.3324/haematol.2020.246603DOI Listing
April 2020

Short-term interleukin-37 treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice.

Aging Cell 2020 01 21;19(1):e13074. Epub 2019 Nov 21.

Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, USA.

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole-body glucose metabolism. Interleukin-37 (IL-37), an anti-inflammatory cytokine of the interleukin-1 family, exerts salutary physiological effects in young mice independent of its inflammation-suppressing properties. Here, we assess the efficacy of IL-37 treatment for improving physiological function in older age. Old mice (26-28 months) received daily intraperitoneal injections of recombinant human IL-37 (recIL-37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10-14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL-37 vs. vehicle-treated mice via increased nitric oxide (NO) bioavailability (all p < .05); this effect was accompanied by enhanced ACh-stimulated NO production and reduced levels of reactive oxygen species in endothelial cells cultured with plasma from IL-37-treated animals (p < .05 vs. vehicle plasma). RecIL-37 treatment increased endurance exercise capacity by 2.4-fold, which was accompanied by a 2.9-fold increase in the phosphorylated AMP-activated kinase (AMPK) to AMPK ratio (i.e., AMPK activation) in quadriceps muscle. RecIL-37 treatment also improved whole-body insulin sensitivity and glucose tolerance (p < .05 vs. vehicle). Improvements in physiological function occurred without significant changes in plasma, aortic, and skeletal muscle pro-inflammatory proteins (under resting conditions), whereas pro-/anti-inflammatory IL-6 was greater in recIL-37-treated animals. Plasma metabolomics analysis revealed that recIL-37 treatment altered metabolites related to pathways involved in NO synthesis (e.g., increased L-arginine and citrulline/arginine ratio) and fatty acid metabolism (e.g., increased pantothenol and free fatty acids). Our findings provide experimental support for IL-37 therapy as a novel strategy to improve diverse physiological functions in old age.
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http://dx.doi.org/10.1111/acel.13074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974720PMC
January 2020

Trisomy 21 activates the kynurenine pathway via increased dosage of interferon receptors.

Nat Commun 2019 10 18;10(1):4766. Epub 2019 Oct 18.

Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Trisomy 21 (T21) causes Down syndrome (DS), affecting immune and neurological function by ill-defined mechanisms. Here we report a large metabolomics study of plasma and cerebrospinal fluid, showing in independent cohorts that people with DS produce elevated levels of kynurenine and quinolinic acid, two tryptophan catabolites with potent immunosuppressive and neurotoxic properties, respectively. Immune cells of people with DS overexpress IDO1, the rate-limiting enzyme in the kynurenine pathway (KP) and a known interferon (IFN)-stimulated gene. Furthermore, the levels of IFN-inducible cytokines positively correlate with KP dysregulation. Using metabolic tracing assays, we show that overexpression of IFN receptors encoded on chromosome 21 contribute to enhanced IFN stimulation, thereby causing IDO1 overexpression and kynurenine overproduction in cells with T21. Finally, a mouse model of DS carrying triplication of IFN receptors exhibits KP dysregulation. Together, our results reveal a mechanism by which T21 could drive immunosuppression and neurotoxicity in DS.
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http://dx.doi.org/10.1038/s41467-019-12739-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800452PMC
October 2019

Transfusional iron overload and intravenous iron infusions modify the mouse gut microbiota similarly to dietary iron.

NPJ Biofilms Microbiomes 2019;5(1):26. Epub 2019 Sep 24.

1Department of Pathology and Cell biology, Columbia University Irving Medical Center, New York, NY USA.

Iron is essential for both microorganisms and their hosts. Although effects of dietary iron on gut microbiota have been described, the effect of systemic iron administration has yet to be explored. Here, we show that dietary iron, intravenous iron administration, and chronic transfusion in mice increase the availability of iron in the gut. These iron interventions have consistent and reproducible effects on the murine gut microbiota; specifically, relative abundance of the and genera negatively correlate with increased iron stores, whereas members of the Clostridia class positively correlate with iron stores regardless of the route of iron administration. Iron levels also affected microbial metabolites, in general, and indoles, in particular, circulating in host plasma and in stool pellets. Taken together, these results suggest that by shifting the balance of the microbiota, clinical interventions that affect iron status have the potential to alter biologically relevant microbial metabolites in the host.
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http://dx.doi.org/10.1038/s41522-019-0097-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760189PMC
June 2020

The metabolic time line of pancreatic cancer: Opportunities to improve early detection of adenocarcinoma.

Am J Surg 2019 12 27;218(6):1206-1212. Epub 2019 Aug 27.

Department of Biochemistry and Molecular Genetics, University of Colorado, USA.

Background: A reliable biomarker to detect pancreatic ductal adenocarcinoma (PDAC) continues to be elusive. With employing metabolomics we hypothesize that a broader analysis of systemic blood can differentiate different stages of PDAC.

Methods: Patients undergoing pancreatic resection had plasma samples grouped by diagnosis and assayed with mass spectrometry. 10 per group [neuroendocrine (PNET), intraductal papillary mucinous neoplasm (IPMN), localized PDAC, locally advanced PDAC, and metastatic] were analyzed to assess if metabolites could delineation different stages of adenocarcinoma.

Results: Of the 215 metabolites measured, four had a stronger correlation to disease burden than CA19-9. However, none of these metabolites differentiated stepwise progression in malignancy. Principal component analysis identified five metabolic components. Each cancer cohort was characterized by a unique combination of components, two components were predictors of PDCA stages.

Conclusions: Enhanced metabolomic analysis identified metabolic pathways that may assist in differentiating PDCA stages that do not occur in a linear stepwise progression.
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http://dx.doi.org/10.1016/j.amjsurg.2019.08.015DOI Listing
December 2019

Differences in Steap3 expression are a mechanism of genetic variation of RBC storage and oxidative damage in mice.

Blood Adv 2019 08;3(15):2272-2285

Bloodworks NW Research Institute, Seattle, WA.

Red blood cells (RBCs) are the most numerous cell type in the body and serve a vital purpose of delivering oxygen to essentially all tissues. In addition to the central role of RBCs in health and disease, RBC storage is a requirement for the >90 million units of RBC transfusions given to millions of recipients each year, worldwide. It is well known that there is genetic donor-to-donor variability in how human RBCs store, rendering blood a nonstandardized therapeutic with a wide range of biological properties from unit to unit, by the time it is transfused. As with humans, genetic variation exists in how murine RBCs, from different strains of mice, store and perform after transfusion. The genetic mechanisms for variation, in humans and mice, both remain obscure. Combining advanced metabolomics, genetics, and molecular and cellular biology approaches, we identify genetic variation in six-transmembrane epithelial antigen of prostate 3 (Steap3) expression as a critical and previously unrecognized mechanism of oxidative damage of RBCs during storage. Increased levels of Steap3 result in degradation of cellular membrane through lipid peroxidation, leading to failure of RBC homeostasis and hemolysis/clearance of RBCs. This article is the first report of a role of Steap3 in mature RBCs; it defines a new mechanism of redox biology in RBCs with a substantial effect upon RBC function and provides a novel mechanistic determinant of genetic variation of RBC storage.
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http://dx.doi.org/10.1182/bloodadvances.2019000605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693009PMC
August 2019

Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

FASEB J 2019 09 29;33(9):10528-10537. Epub 2019 Jun 29.

Department of Biochemistry and Molecular Biology, The University of Texas McGovern Medical School, Houston, Texas, USA.

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls. Given the vital role of period 2 (Per2) in the core clock and the unrecognized role of Per2 in SCD, we transplanted the bone marrow (BM) of SCD mice to mice, which revealed that Per2 expression was up-regulated in SCD mouse lung. Next, we transplanted the BM of SCD mice to period 1 () [ ()] and wild-type mice, respectively. We discovered that mice transplanted with SCD BM (SCD → ) displayed severe irradiation sensitivity and were more susceptible to an early death. Although we observed an increase of peripheral inflammatory cells, we did not detect differences in erythrocyte sickling. However, there was further lung damage due to elevated pulmonary congestion, inflammatory cell infiltration, iron overload, and secretion of IL-6 in lavage fluid. Overall, we demonstrate that is beneficial to counteract elevated systemic inflammation, lung tissue inflammation, and iron overload in SCD.-Adebiyi, M. G., Zhao, Z., Ye, Y., Manalo, J., Hong, Y., Lee, C. C., Xian, W., McKeon, F., Culp-Hill, R., D' Alessandro, A., Kellems, R. E., Yoo, S.-H., Han, L., Xia, Y. Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.
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http://dx.doi.org/10.1096/fj.201900246RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988849PMC
September 2019

Cysteine depletion targets leukemia stem cells through inhibition of electron transport complex II.

Blood 2019 07 17;134(4):389-394. Epub 2019 May 17.

Division of Hematology and.

We have previously demonstrated that oxidative phosphorylation is required for the survival of human leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). More recently, we demonstrated that LSCs in patients with de novo AML rely on amino acid metabolism to drive oxidative phosphorylation. Notably, although overall levels of amino acids contribute to LSC energy metabolism, our current findings suggest that cysteine may be of particular importance for LSC survival. We demonstrate that exogenous cysteine is metabolized exclusively to glutathione. Upon cysteine depletion, glutathione synthesis is impaired, leading to reduced glutathionylation of succinate dehydrogenase A (SDHA), a key component of electron transport chain complex (ETC) II. Loss of SDHA glutathionylation impairs ETC II activity, thereby inhibiting oxidative phosphorylation, reducing production of ATP, and leading to LSC death. Given the role of cysteine in driving LSC energy production, we tested cysteine depletion as a potential therapeutic strategy. Using a novel cysteine-degrading enzyme, we demonstrate selective eradication of LSCs, with no detectable effect on normal hematopoietic stem/progenitor cells. Together, these findings indicate that LSCs are aberrantly reliant on cysteine to sustain energy metabolism, and that targeting this axis may represent a useful therapeutic strategy.
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http://dx.doi.org/10.1182/blood.2019898114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659257PMC
July 2019

The TP53 Apoptotic Network Is a Primary Mediator of Resistance to BCL2 Inhibition in AML Cells.

Cancer Discov 2019 07 2;9(7):910-925. Epub 2019 May 2.

Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.

To study mechanisms underlying resistance to the BCL2 inhibitor venetoclax in acute myeloid leukemia (AML), we used a genome-wide CRISPR/Cas9 screen to identify gene knockouts resulting in drug resistance. We validated , , and as genes whose inactivation results in venetoclax resistance in AML cell lines. Resistance to venetoclax resulted from an inability to execute apoptosis driven by BAX loss, decreased expression of BCL2, and/or reliance on alternative BCL2 family members such as BCL2L1. The resistance was accompanied by changes in mitochondrial homeostasis and cellular metabolism. Evaluation of knockout cells for sensitivities to a panel of small-molecule inhibitors revealed a gain of sensitivity to TRK inhibitors. We relate these observations to patient drug responses and gene expression in the Beat AML dataset. Our results implicate , the apoptotic network, and mitochondrial functionality as drivers of venetoclax response in AML and suggest strategies to overcome resistance. SIGNIFICANCE: AML is challenging to treat due to its heterogeneity, and single-agent therapies have universally failed, prompting a need for innovative drug combinations. We used a genetic approach to identify genes whose inactivation contributes to drug resistance as a means of forming preferred drug combinations to improve AML treatment...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606338PMC
July 2019

Quantitative metabolomics comparison of traditional blood draws and TAP capillary blood collection.

Metabolomics 2018 07 12;14(7):100. Epub 2018 Jul 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, 80045, USA.

Introduction: Mass spectrometry and computational biology have advanced significantly in the past ten years, bringing the field of metabolomics a step closer to personalized medicine applications. Despite these analytical advancements, collection of blood samples for routine clinical analysis is still performed through traditional blood draws.

Objective: TAP capillary blood collection has been recently introduced for the rapid, painless draw of small volumes of blood (~ 100 μL), though little is known about the comparability of metabolic phenotypes of blood drawn via traditional venipuncture and TAP devices.

Methods: UHPLC-MS-targeted metabolomics analyses were performed on blood drawn traditionally or through TAP devices from 5 healthy volunteers. Absolute quantitation of 45 clinically-relevant metabolites was calculated against stable heavy isotope-labeled internal standards.

Results: Ranges for 39 out of 45 quantified metabolites overlapped between drawing methods. Pyruvate and succinate were over threefold higher in the TAP samples than in traditional blood draws. No significant changes were observed for other carboxylates, glucose or lactate. TAP samples were characterized by increases in reduced glutathione and decreases in urate and cystine, markers of oxidation of purines and cysteine-overall suggesting decreased oxidation during draws. The absolute levels of bile acids and acyl-carnitines, as well as almost all amino acids, perfectly correlated among groups (Spearman r ≥ 0.95).

Conclusion: Though further more extensive studies will be mandatory, this pilot suggests that TAP-derived blood may be a logistically-friendly source of blood for large scale metabolomics studies-especially those addressing amino acids, glycemia and lactatemia as well as bile acids, acyl-carnitine levels.
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http://dx.doi.org/10.1007/s11306-018-1395-zDOI Listing
July 2018

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

Cell 2019 01 20;176(3):564-580.e19. Epub 2018 Dec 20.

Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Electronic address:

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.
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http://dx.doi.org/10.1016/j.cell.2018.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483089PMC
January 2019

Inhibition of Amino Acid Metabolism Selectively Targets Human Leukemia Stem Cells.

Cancer Cell 2018 11;34(5):724-740.e4

Division of Hematology, University of Colorado Denver, Anschutz Medical Campus, 12700 East 19(th) Avenue, Aurora, CO 80045, USA. Electronic address:

In this study we interrogated the metabolome of human acute myeloid leukemia (AML) stem cells to elucidate properties relevant to therapeutic intervention. We demonstrate that amino acid uptake, steady-state levels, and catabolism are all elevated in the leukemia stem cell (LSC) population. Furthermore, LSCs isolated from de novo AML patients are uniquely reliant on amino acid metabolism for oxidative phosphorylation and survival. Pharmacological inhibition of amino acid metabolism reduces oxidative phosphorylation and induces cell death. In contrast, LSCs obtained from relapsed AML patients are not reliant on amino acid metabolism due to their ability to compensate through increased fatty acid metabolism. These findings indicate that clinically relevant eradication of LSCs can be achieved with drugs that target LSC metabolic vulnerabilities.
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http://dx.doi.org/10.1016/j.ccell.2018.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280965PMC
November 2018

Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia.

Nat Med 2018 12 12;24(12):1859-1866. Epub 2018 Nov 12.

Division of Hematology, University of Colorado School of Medicine, Aurora, CO, USA.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse. In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes.
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http://dx.doi.org/10.1038/s41591-018-0233-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001730PMC
December 2018

Heterogeneity of blood processing and storage additives in different centers impacts stored red blood cell metabolism as much as storage time: lessons from REDS-III-Omics.

Transfusion 2019 01 24;59(1):89-100. Epub 2018 Oct 24.

BloodWorks Northwest, Seattle, Washington.

Background: Biological and technical variability has been increasingly appreciated as a key factor impacting red blood cell (RBC) storability and, potentially, transfusion outcomes. Here, we performed metabolomics analyses to investigate the impact of factors other than storage duration on the metabolic phenotypes of stored RBC in a multicenter study.

Study Design And Methods: Within the framework of the REDS-III (Recipient Epidemiology and Donor Evaluation Study-III) RBC-Omics study, 13,403 donors were enrolled from four blood centers across the United States and tested for the propensity of their RBCs to hemolyze after 42 days of storage. Extreme hemolyzers were recalled and donated a second unit of blood. Units were stored for 10, 23, and 42 days prior to sample acquisition for metabolomics analyses.

Results: Unsupervised analyses of metabolomics data from 599 selected samples revealed a strong impact (14.2% of variance) of storage duration on metabolic phenotypes of RBCs. The blood center collecting and processing the units explained an additional 12.2% of the total variance, a difference primarily attributable to the storage additive (additive solution 1 vs. additive solution 3) used in the different hubs. Samples stored in mannitol-free/citrate-loaded AS-3 were characterized by elevated levels of high-energy compounds, improved glycolysis, and glutathione homeostasis. Increased methionine metabolism and activation of the transsulfuration pathway was noted in samples processed in the center using additive solution 1.

Conclusion: Blood processing impacts the metabolic heterogeneity of stored RBCs from the largest multicenter metabolomics study in transfusion medicine to date. Studies are needed to understand if these metabolic differences influenced by processing/storage strategies impact the effectiveness of transfusions clinically.
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http://dx.doi.org/10.1111/trf.14979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322946PMC
January 2019

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells.

Transfusion 2018 12 12;58(12):2978-2991. Epub 2018 Oct 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.

Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank.

Study Design And Methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway.

Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3.

Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.
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http://dx.doi.org/10.1111/trf.14936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357231PMC
December 2018

Effects of red blood cell (RBC) transfusion on sickle cell disease recipient plasma and RBC metabolism.

Transfusion 2018 12 28;58(12):2797-2806. Epub 2018 Sep 28.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.

Background: Exchange transfusion is a mainstay in the treatment of sickle cell anemia. Transfusion recipients with sickle cell disease (SCD) can be transfused over 10 units per therapy, an intervention that replaces circulating sickle red blood cells (RBCs) with donor RBCs. Storage of RBCs makes the intervention logistically feasible. The average storage duration for units transfused at the Duke University Medical Center is approximately 2 weeks, a time window that should anticipate the accumulation of irreversible storage lesion to the RBCs. However, no metabolomics study has been performed to date to investigate the impact of exchange transfusion on recipients' plasma and RBC phenotypes.

Study Design And Methods: Plasma and RBCs were collected from patients with sickle cell anemia before transfusion and within 5 hours after exchange transfusion with up to 11 units, prior to metabolomics analyses.

Results: Exchange transfusion significantly decreased plasma levels of markers of systemic hypoxemia like lactate, succinate, sphingosine 1-phosphate, and 2-hydroxyglutarate. These metabolites accumulated in transfused RBCs, suggesting that RBCs may act as scavenger/reservoirs. Transfused RBCs displayed higher glycolysis, total adenylate pools, and 2,3-diphosphoglycerate, consistent with increased capacity to deliver oxygen. Plasma levels of acyl-carnitines and amino acids decreased, while fatty acids and potentially harmful phthalates increased upon exchange transfusion.

Conclusion: Metabolic phenotypes confirm the benefits of the transfusion therapy in transfusion recipients with SCD and the reversibility of some of the metabolic storage lesion upon transfusion in vivo in 2-week-old RBCs. However, results also suggest that potentially harmful plasticizers are transfused.
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http://dx.doi.org/10.1111/trf.14931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283695PMC
December 2018

A Role for Tryptophan-2,3-dioxygenase in CD8 T-cell Suppression and Evidence of Tryptophan Catabolism in Breast Cancer Patient Plasma.

Mol Cancer Res 2019 01 24;17(1):131-139. Epub 2018 Aug 24.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Tryptophan catabolism is an attractive target for reducing tumor progression and improving antitumor immunity in multiple cancers. Tumor infiltration by CD8 T cells correlates with improved prognosis in triple-negative breast cancer (TNBC) and a significant effort is underway to improve CD8 T-cell antitumor activity. In this study, primary human immune cells were isolated from the peripheral blood of patients and used to demonstrate that the tryptophan catabolite kynurenine induces CD8 T-cell death. Furthermore, it is demonstrated that anchorage-independent TNBC utilizes the tryptophan-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) to inhibit CD8 T-cell viability. Publicly available data revealed that high , the gene encoding TDO, correlates with poor breast cancer clinical outcomes, including overall survival and distant metastasis-free survival, while expression of the gene encoding the more commonly studied tryptophan-catabolizing enzyme, did not. Metabolomic analysis, using quantitative mass spectrometry, of tryptophan and its catabolites, including kynurenine, in the plasma from presurgical breast cancer patients ( = 77) and 40 cancer-free donors ( = 40) indicated a strong correlation between substrate and catabolite in both groups. Interestingly, both tryptophan and kynurenine were lower in the plasma from patients with breast cancer compared with controls, particularly in women with estrogen receptor (ER)-negative and stage III and IV breast cancer. IMPLICATIONS: This study underscores the importance of tryptophan catabolism, particularly in aggressive disease, and suggests that future pharmacologic efforts should focus on developing drugs that target both TDO and IDO1.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318037PMC
January 2019

Metabolomics evaluation of early-storage red blood cell rejuvenation at 4°C and 37°C.

Transfusion 2018 08 24;58(8):1980-1991. Epub 2018 Apr 24.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.

Background: Refrigerated red blood cell (RBC) storage results in the progressive accumulation of biochemical and morphological alterations collectively referred to as the storage lesion. Storage-induced metabolic alterations can be in part reversed by rejuvenation practices. However, rejuvenation requires an incubation step of RBCs for 1 hour at 37°C, limiting the practicality of providing "on-demand," rejuvenated RBCs. We tested the hypothesis that the addition of rejuvenation solution early in storage as an adjunct additive solution would prevent-in a time window consistent with the average age of units transfused to sickle cell recipients at Duke (15 days)-many of the adverse biochemical changes that can be reversed via standard rejuvenation, while obviating the incubation step.

Study Design And Methods: Metabolomics analyses were performed on cells and supernatants from AS-1 RBC units (n = 4), stored for 15 days. Units were split into pediatric bag aliquots and stored at 4°C. These were untreated controls, washed with or without rejuvenation, performed under either standard (37°C) or cold (4°C) conditions.

Results: All three treatments removed most metabolic storage by-products from RBC supernatants. However, only standard and cold rejuvenation provided significant metabolic benefits as judged by the reactivation of glycolysis and regeneration of adenosine triphosphate and 2,3-diphosphoglycerate. Improvements in energy metabolism also translated into increased capacity to restore the total glutathione pool and regenerate oxidized vitamin C in its reduced (ascorbate) form.

Conclusion: Cold and standard rejuvenation of 15-day-old RBCs primes energy and redox metabolism of stored RBCs, while providing a logistic advantage for routine blood bank processing workflows.
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http://dx.doi.org/10.1111/trf.14623DOI Listing
August 2018

Metabolic effect of alkaline additives and guanosine/gluconate in storage solutions for red blood cells.

Transfusion 2018 08 6;58(8):1992-2002. Epub 2018 Apr 6.

Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, the Netherlands.

Background: Over a century of advancements in the field of additive solutions for red blood cell (RBC) storage has made transfusion therapy a safe and effective practice for millions of recipients worldwide. Still, storage in the blood bank results in the progressive accumulation of metabolic alterations, a phenomenon that is mitigated by storage in novel storage additives, such as alkaline additive solutions. While novel alkaline additive formulations have been proposed, no metabolomics characterization has been performed to date.

Study Design And Methods: We performed UHPLC-MS metabolomics analyses of red blood cells stored in SAGM (standard additive in Europe), (PAGGSM), or alkaline additives SOLX, E-SOL 5 and PAG3M for either 1, 21, 35 (end of shelf-life in the Netherlands), or 56 days.

Results: Alkaline additives (especially PAG3M) better preserved 2,3-diphosphoglycerate and adenosine triphosphate (ATP). Deaminated purines such as hypoxanthine were predictive of hemolysis and morphological alterations. Guanosine supplementation in PAGGSM and PAG3M fueled ATP generation by feeding into the nonoxidative pentose phosphate pathway via phosphoribolysis. Decreased urate to hypoxanthine ratios were observed in alkaline additives, suggestive of decreased generation of urate and hydrogen peroxide. Despite the many benefits observed in purine and redox metabolism, alkaline additives did not prevent accumulation of free fatty acids and oxidized byproducts, opening a window for future alkaline formulations including (lipophilic) antioxidants.

Conclusion: Alkalinization via different strategies (replacement of chloride anions with either high bicarbonate, high citrate/phosphate, or membrane impermeant gluconate) results in different metabolic outcomes, which are superior to current canonical additives in all cases.
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http://dx.doi.org/10.1111/trf.14620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131048PMC
August 2018

Red blood cell metabolism in Down syndrome: hints on metabolic derangements in aging.

Blood Adv 2017 Dec 21;1(27):2776-2780. Epub 2017 Dec 21.

Department of Biochemistry and Molecular Genetics.

Red blood cells (RBCs) are the most abundant cell in the human body. During their ∼120-day life span in the circulatory system, RBCs release oxygen to all human tissues while being exposed to tissue metabolic activity. Owing to the relative simplicity of their intrinsic metabolism and the abundance of metabolite transporters in RBC membranes, the metabolism of mature erythrocytes indirectly mirrors systemic metabolic homeostasis and its alterations as a function of physiological factors, such as aging. Trisomy 21 (T21), the etiological factor of Down syndrome (DS), has been shown to cause chronic autoinflammation, promoting alterations in RBC life span, size (macrocytosis), and redox homeostasis. Here, we provide the first mass spectrometry-based relative and absolute quantitative metabolomic description of human RBCs from volunteer disomic and trisomic donors (n = 97). The results indicate a widespread deregulation of T21 RBC metabolism, including significant intracellular accumulation of lactate, amino acids (except methionine), purine catabolites, glutathione metabolites, carboxylic acids, bile acids (especially conjugated ones), and acyl-conjugated carnitines. These changes may underlie some of the well-established comorbidities in DS. Finally, we identify sex- and/or T21-specific metabolic signatures of aging.
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http://dx.doi.org/10.1182/bloodadvances.2017011957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745140PMC
December 2017

Red blood cells in hemorrhagic shock: a critical role for glutaminolysis in fueling alanine transamination in rats.

Blood Adv 2017 Jul 14;1(17):1296-1305. Epub 2017 Jul 14.

Department of Biochemistry and Molecular Genetics.

Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography-mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo-injected CN-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage.
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http://dx.doi.org/10.1182/bloodadvances.2017007187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727973PMC
July 2017

Investigation of the effects of storage and freezing on mixes of heavy-labeled metabolite and amino acid standards.

Rapid Commun Mass Spectrom 2017 Dec;31(23):2030-2034

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, 12801 East 17th Ave, Aurora, CO, 80045, USA.

Rationale: High-throughput metabolomics has now made it possible for small/medium-sized laboratories to analyze thousands of samples/year from the most diverse biological matrices including biofluids, cell and tissue extracts. In large-scale metabolomics studies, stable-isotope-labeled standards are increasingly used to normalize for matrix effects and control for technical reproducibility (e.g. extraction efficiency, chromatographic retention times and mass spectrometry signal stability). However, it is currently unknown how stable mixes of commercially available standards are following repeated freeze/thaw cycles or prolonged storage of aliquots.

Methods: Standard mixes for C, N or deuterated isotopologues of amino acids and key metabolites from the central carbon and nitrogen pathways (e.g. glycolysis, Krebs cycle, redox homeostasis, purines) were either repeatedly frozen/thawed for up to 10 cycles or diluted into aliquots prior to frozen storage for up to 42 days. Samples were characterized by ultra-high-pressure liquid chromatography/mass spectrometry to determine the stability of the aliquoted standards upon freezing/thawing or prolonged storage.

Results: Metabolite standards were stable over up to 10 freeze/thaw cycles, with the exception of adenosine and glutathione, showing technical variability across aliquots in a freeze/thaw-cycle-independent fashion. Storage for up to 42 days of mixes of commercially available standards did not significantly affect the stability of amino acid or metabolite standards for the first 2 weeks, while progressive degradation (statistically significant for fumarate) was observed after 3 weeks.

Conclusions: Refrigerated or frozen preservation for at least 2 weeks of aliquoted heavy-labeled standard mixes for metabolomics analysis is a feasible and time-/resource-saving strategy for standard metabolomics laboratories.
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http://dx.doi.org/10.1002/rcm.7989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673535PMC
December 2017