Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma

Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma Sunada Khadka, Kenisha Arthur, Mykia Washington, Yasaman Barekatain, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-125147/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates by oxidation of anaplerotic substrates, such as glucose-derived pyruvate and glutamine20 derived glutamate. We have previously documented that a subset of tumors with 1p36 homozygous deletion exhibit co-deletion of ENO1, in turn becoming extremely dependent on its redundant isoform ENO2 and sensitive to an overall enzymatic deficiency of enolase. Metabolomic profiling of ENO1-deleted glioma cells treated with an enolase inhibitor revealed a profound decrease in TCA cycle metabolites, which correlated with cell-line specific sensitivity to enolase inhibition, highlighting the importance of glycolysis derived pyruvate for anaplerosis. Correspondingly, the toxicity of the enolase inhibitor was significantly attenuated by exogenous supplementation of supraphysiological levels of anaplerotic substrates including pyruvate. These findings led us to hypothesize that cancer cells with ENO1 homozygous deletions treated with an enolase inhibitor might show exceptional sensitivity to inhibition of glutaminolysis because of reduced anaplerotic flow from glycolysis. We found that ENO1-deleted cells indeed exhibited selective sensitivity to the glutaminase inhibitor CB-839, and this sensitivity was also attenuated by exogenous supplementation of anaplerotic substrates including pyruvate. Despite these promising in vitro results, the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for 1p36-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of the in vitro and in vivo environments as the potential reasons for the differential efficacy of metabolism targeted therapies in in vitro and in vivo systems. Cancer Biology Cancer Metabolism Anaplerosis Collateral Lethality Glycolysis Glutaminolysis Enolase Inhibitor POMHEX CB-839 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Tables Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Supplementary Files SupplmentaryFigures1and2.pdf SupplementalFigures.pdf Table1.JPG representing the IC 50 values of ENO 1 deleted cells treated with POMHEX in pyruvate free DMEM or DMEM exogenously supplemented with different anaplerotic substrates at the indicated concentrations IC 50 values in red indicate metabolites that confer partial or complete rescue to POMHEX toxicity Human plasma metabolite concentrations are indicated where relevant, based on human metabolome database ( Pyruvic acid (HMDB 0000243 lactic acid (HMDB 0000190 Acetate,Acetate,(HMDB 0000042 Alanine, (HMDB 0000161 Aspartate HMDB 0000191 19 Oxovalerate (HMDB 0001865 Geigy Scientific Tables, 8 th Rev edition, pp 165 177 Edited by Cornelius Lentner Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 10 Jan, 2021 Editorial decision: Major revision 10 Jan, 2021 Review # 2 received at journal 05 Jan, 2021 Reviewer # 2 agreed at journal 15 Dec, 2020 Reviewer # 1 agreed at journal 14 Dec, 2020 Reviewers invited by journal 13 Dec, 2020 First submitted to journal 06 Dec, 2020 Editor assigned by journal 06 Dec, 2020 Submission checks completed at journal 06 Dec, 2020 Editor invited by journal 06 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-125147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":6149456,"identity":"c9c081d1-8ffb-44a0-a3c6-4ed3bb18fafe","order_by":0,"name":"Sunada Khadka","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sunada","middleName":"","lastName":"Khadka","suffix":""},{"id":6149457,"identity":"f276400f-0f8a-4b7f-813e-22bc746f2144","order_by":1,"name":"Kenisha Arthur","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kenisha","middleName":"","lastName":"Arthur","suffix":""},{"id":6149458,"identity":"c6098280-ffe0-4ef3-9438-c1502e581cfb","order_by":2,"name":"Mykia Washington","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mykia","middleName":"","lastName":"Washington","suffix":""},{"id":6149459,"identity":"b9637987-41e6-4968-a26d-4ccee5a4b882","order_by":3,"name":"Yasaman Barekatain","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasaman","middleName":"","lastName":"Barekatain","suffix":""},{"id":6149460,"identity":"241aa9a0-323d-492f-b53d-7877bad805bc","order_by":4,"name":"Jeff Ackroyd","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeff","middleName":"","lastName":"Ackroyd","suffix":""},{"id":6149461,"identity":"2faafb24-2e12-4e2c-99bd-b9b04b88ace0","order_by":5,"name":"Eliot Behr","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eliot","middleName":"","lastName":"Behr","suffix":""},{"id":6149462,"identity":"cce4fce7-6c2d-491b-aafb-624a486a3165","order_by":6,"name":"Pornpa Suriyamongkol","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pornpa","middleName":"","lastName":"Suriyamongkol","suffix":""},{"id":6149463,"identity":"a8ceffaf-78ea-4d76-8a7d-79d651c33680","order_by":7,"name":"Yu-Hsi Lin","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Hsi","middleName":"","lastName":"Lin","suffix":""},{"id":6149464,"identity":"51cc062e-7af5-4508-8c7d-f3cec073340a","order_by":8,"name":"Kaitlyn Crowley","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaitlyn","middleName":"","lastName":"Crowley","suffix":""},{"id":6149465,"identity":"53609171-0916-4a22-bbd4-a4ac372bfe2f","order_by":9,"name":"Cong-Dat Pham","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cong-Dat","middleName":"","lastName":"Pham","suffix":""},{"id":6149466,"identity":"a4f11c20-fcba-4c31-8739-1dad6c8e0ca8","order_by":10,"name":"Dimitra K. Georgiou","email":"","orcid":"","institution":"The University of Texas MD Anderson Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dimitra","middleName":"K.","lastName":"Georgiou","suffix":""},{"id":6149467,"identity":"36b7f85a-f751-4547-9911-9f6d55d0daf6","order_by":11,"name":"John Asara","email":"","orcid":"","institution":"Beth Israel Deaconess Medical Center and Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Asara","suffix":""},{"id":6149468,"identity":"914da1bc-335e-40df-b13a-745d95648011","order_by":12,"name":"Florian 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15:29:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-125147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-125147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4286714,"identity":"3c49a08f-e209-4179-b874-d5ca3096a621","added_by":"auto","created_at":"2020-12-15 21:24:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":210700,"visible":true,"origin":"","legend":"Metabolomic profiling of Enolase inhibitor treated glioma cell lines indicates a profound disruption in anaplerosis, which correlates with the sensitivity A C Sensitivity of glioma cells to the enolase inhibitor is reflected by their ENO 1 status A Dose response curves of ENO 1 homozygously deleted (D 423 red, N= 4 ENO 1 heterozygously deleted (D 502 green, N= 4 U 343 orange, N= 4 and ENO 1 wild type (LN 319 grey N= 4 cells treated with the enolase inhibitor POMHEX at the indicated\ndoses Error bars represent standard error of mean B After 5 days of treatment, the cells were fixed in 10 formalin and stained with crystal violet dye to measure the terminal cell density The terminal cell density is expressed relative to the untreated controls C A representative table with the IC 50 values of POMHEX across different cell lines strongly indicates that ENO 1 homozygously deleted cells are selectively sensitive, while ENO 1 heterozygotes display intermediate sensitivity to POMHEX D E Enolase inhibitor causes a profound disruption in the TCA cycle Cells were treated with varying concentrations of POMHEX in DMEM media and the metabolites were extracted in 80 cold methanol after 72 hours of drug treatment The extracted metabolites were subjected to metabolomic analysis by mass spectroscopy D Schematic showing the glycolytic and TCA cycle metabolites that are altered by POMHEX treatment E Lactate levels are shown as an indicator of glycolysis inhibition in response to the enolase inhibitor POMHEX Two TCA cycle intermediates citrate and malate are shown as representative metabolites in the TCA cycle that are altered in a dose dependent manner as a result of enolase inhibition The effects of enolase inhibition on TCA cycle metabolites correlate with the levels of ENO 1 in different cell lines, with the ENO 1 homozygously deleted cells exhibiting the most profound change, followed by ENO 1 heterozygous cells showing intermediate effect while ENO 1 intact wild type cells sustaining no significant effect ..(See supplemental figure S 1 and S 2 a full panel of glycolytic and TCA cycle metabolites","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/6b49fe0b75804606eb46635c.jpg"},{"id":4286717,"identity":"a5571abf-c9b1-468f-be7f-d4c3631674f2","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99679,"visible":true,"origin":"","legend":"Exogenous supplementation of selected anaplerotic substrates mitigates the toxicity of enolase inhibition A Schematic representing different cellular metabolites that converge to replenish TCA cycle carbon atoms Exogenously supplemented anaplerotic substrates ( acetate, aspartate, fatty acids, lactate, methylpyruvate pyruvate, oxaloacetate, and oxovalerate are indicated in blue, while cataplerotic substrates are indicated in red B Dose response curves of ENO 1 deleted, ENO 1 rescued and ENO 1 wild type cells to POMHEX in pyruvate free medium ( 12 and medium exogenously supplemented with 5 mM pyruvate ( 6 and 2 5 mM methyl pyruvate ( 4 Cells were\nseeded in 96 wells plates in pyruvate free DMEM or DMEM supplemented with anaplerotic substrates and treated with serial dilutions of POMHEX Crystal violet staining was performed to measure the terminal cell density and assess the effect of POMHEX and the degree of rescue of POMHEX toxicity by exogenous supplementation of anaplerotic substrates Cell density is expressed relative to the\nuntreated controls A shift in IC 50 indicates alleviation of POMHEX toxicity by addition of exogenous anaplerotic substrates IC 50 of POMHEX for each cell line in different medium condition is indicated (See Supplemental Figure S 3 for a panel of anaplerotic substrates)","description":"","filename":"Fig2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/570d68a93333c773ca0b0221.JPG"},{"id":4286720,"identity":"24846c81-53c8-4f08-af69-7a3b2652265a","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153054,"visible":true,"origin":"","legend":"Media pyruvate availability modulates sensitivity to glutaminase inhibitor A Cells were treated with 500 nM CB 839 in either pyruvate free DMEM or DMEM supplemented with 5 mM pyruvate for 5 days Crystal violet staining was performed to determine the effect of CB 839 on cell growth Cell density is expressed relative to vehicle control in pyruvate free medium ENO 1 deleted N= 16 ENO 1 rescued N= 16 and ENO 1 WT N= 16 Mean and S D are shown Where indicated, represent statistical significance ( 0 0001 determined by 2 way ANOVA and Tukey’s post hoc analysis B Metabolic map representing the intermediates in the central carbon metabolic pathways CB 839 targets glutaminase, the enzyme that converts glutamine to glutamate, and impedes glutamate anaplerosis to the TCA cycle C,D Metabolomics analysis reveal TCA cycle intermediate depletion as a major consequence of CB 839 treatment Cells were treated with 500 nM CB 839 in pyruvate free or regular DMEM 1 mM Pyruvate) for 72 hours and metabolites were extracted in 80 cold methanol and metabolite abundance was determined by MS Representative TCA cycle metabolites that are altered into CB 839 treatment in pyruvate free C and pyruvate replete DMEM D","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/97a889ca707914dda231fcd3.jpg"},{"id":4286721,"identity":"5f14e49d-0690-4e83-97d5-890391949c00","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161393,"visible":true,"origin":"","legend":"Synergistic anti neoplastic effect of POMHEX and CB 839 in pyruvate free condition ENO 1 homozygously deleted (D 423 red, N= 2 ENO 1 isogenically rescued (D 423 ENO 1 blue, N= 2 and ENO 1 WT (LN 319 grey, N= 2 cells were seeded in 96 well plates After 24 hours, the cells were treated with serial dilutions of POMHEX alone A and B or in combination with a fixed 500 nM CB 839 in pyruvate free and pyruvate supplemented medium Columns 1 2 vehicle control 3 10 serial dilutions of POMHEX and constant 500 nM CB 839 11 12 constant 500 nM CB 839 C and D The cells were grown in pyruvate free A and C or 5 mM pyruvate supplemented medium B and D Following 5 days of drug treatment, cells were fixed and crystal violet staining was performed to determine cell density in response to the drug treatment Data are expressed relative to the untreated control Note the substantial synergy between POMHEX and CB 839 which is accentuated in pyruvate free condition C and partially reversed by pyruvate supplementation D E Schematic showing the inhibition of glycolysis by POMHEX at the enolase step, and inhibition of glutaminolysis by CB 839 both converging to impede TCA cycle anaplerosis at different steps of the cycle Combined inhibition of glycolysis and glutaminolysis especially under pyruvate free conditions could synergistically deplete TCA cycle intermediates and induce dramatic cell death","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/ba5ae3badb6fbfb39ecaf2c9.jpg"},{"id":4286722,"identity":"4b9737b2-9e85-4dc3-9df1-f63c04060bd8","added_by":"auto","created_at":"2020-12-15 21:24:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91233,"visible":true,"origin":"","legend":"CB 839 and HEX combination attenuates intracranial tumor growth but does not cause a frank tumor regression ENO 1 deleted glioma cells (D 423 were implanted intracranially in immunocompromised nude mice and tumor growth was monitored weekly by T 2 MRI Tumors are MRI detectable (indicated by dashed yellow outlines) 20 30 days after tumor implantation 3 D Slicer was used to view the DICOM files and measure tumor volumes A Representative MRI images to indicate weekly changes in tumor volume across different treatment groups, Control ( 2 CB 839 treated 200 mpk BID orally N= 2 HEX treated 300 mpk SC N= 2 CB 839 +HEX 200 mpk CB 839 BID orally,\nand 300 mpk HEX SC N= 2 Following the completion of treatment course, animals were sacrificed, and the brains were dissected and fixed in formaldehyde for histopathological analyses B Pre and post treatment comparison of absolute and relative tumor volumes across different treatment groups C Percent change in tumor volume after two weeks of drug treatment","description":"","filename":"Fig5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/d7a2f36f57d5fb5104c0705e.JPG"},{"id":4286723,"identity":"20618fbe-727a-4ee9-b4dc-2eda9fe32d6c","added_by":"auto","created_at":"2020-12-15 21:24:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":164390,"visible":true,"origin":"","legend":"Glutaminase inhibition does not show anti tumor activity against ENO1 deleted subcutaneous tumors. ENO 1 deleted glioma cells (D 423 were implanted subcutaneously in immunocompromised nude mice and once the tumors reached 200 mm 3 mice were randomly assigned into different treatment groups Vehicle\ncontrol ( 3 CB 839 200 mpk BID orally N= 3 HEX 300 mpk SC N= 3 CB 839 +HEX 200 mpk CB 839 BID orally, and 300 mpk HEX SC N= 3 A B Tumor volume changes in response to the drug treatment was determined by measuring tumors three times a week using Vernier’s calipers A Absolute tumor volume (mm 3 and B Relative tumor volume growth curves during one month treatment course Following the completion of treatment course, animals were sacrificed, and the tumors were dissected and fixed in formalin for histopathological analyses or frozen in liquid nitrogen for metabolomic analyses C D IHC staining for cell proliferation marker (phospho histone 3 ,pH 3 black stain, blue arrows) and marker of apoptosis (cleaved caspase 3 CC 3 black stain, red arrows) in tissue sections of control, CB 839 HEX and CB 839 HEX treated tumors size bar, 300 µm D Counts of p H 3 and CC 3 positive cells per 100 X section are shown E F Metabolomic analysis of frozen tumors show key differences in metabolites upstream and downstream of enolase reaction in HEX treated tumors Tumors were extracted approximately 4 6 hours\nafter the final dose Representative glycolytic intermediates (top panel, E and TCA cycle intermediates (bottom panel, F altered in response to drug treatments Metabolites are expressed relative to the vehicle control ..( N= 3 CB 839 N= 3 HEX, N= 1 and CB 839 HEX, N= 3 Mean and S D where relevant with individual data points are shown) Where indicated, represents statistical significance ( 0 05\nachieved by 2 way ANOVA and Tukey’s post hoc analysis ..(Metabolomics data represented in this panel were obtained using metabolomics core at BIDMC and included N= 1 HEX treated tumor Remaining HEX treated tumors from this experiment were used for metabolomics with the Metabolon Inc platform See\nsupplemental Figure S 7 ","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/08734ef25fa19223fae3bc6e.jpg"},{"id":13634082,"identity":"444c06ce-ffae-4c62-8725-b0c21e601229","added_by":"auto","created_at":"2021-09-17 08:30:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":739620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/7fdaa963-03e2-48be-a9d2-b1ef543fefe9.pdf"},{"id":4286716,"identity":"7d100f45-bb9a-4768-b8e2-99236a8133e7","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":771431,"visible":true,"origin":"","legend":"","description":"","filename":"SupplmentaryFigures1and2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/be499940615fe215dacd55c6.pdf"},{"id":4286718,"identity":"562ac96e-89c0-468b-9a02-846e5e9ddcfe","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1638317,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/9ced72fae0072f66eeea8fa1.pdf"},{"id":4286719,"identity":"fa357f4d-7da7-4caf-98ac-1bbfb048ea42","added_by":"auto","created_at":"2020-12-15 21:24:26","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32522,"visible":true,"origin":"","legend":"representing the IC 50 values of ENO 1 deleted cells treated with POMHEX in pyruvate free DMEM or DMEM exogenously supplemented with different anaplerotic substrates at the indicated concentrations IC 50 values in red indicate metabolites that confer partial or complete rescue to POMHEX toxicity Human plasma metabolite concentrations are indicated where relevant, based on human metabolome database (\nPyruvic acid (HMDB 0000243 lactic acid (HMDB 0000190 Acetate,Acetate,(HMDB 0000042 Alanine, (HMDB 0000161 Aspartate HMDB 0000191 19 Oxovalerate (HMDB 0001865 Geigy Scientific Tables, 8 th Rev edition, pp 165 177 Edited by Cornelius Lentner","description":"","filename":"Table1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-125147/v1/d10fb32eb1f8853bab315b03.JPG"}],"financialInterests":"","formattedTitle":"\u003cp\u003eImpaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-125147/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eDue to technical limitations, table 1 is only available as a download in the Supplemental Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer Metabolism, Anaplerosis, Collateral Lethality, Glycolysis, Glutaminolysis, Enolase Inhibitor, POMHEX, CB-839","lastPublishedDoi":"10.21203/rs.3.rs-125147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-125147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates by oxidation of anaplerotic substrates, such as glucose-derived pyruvate and glutamine20 derived glutamate. We have previously documented that a subset of tumors with 1p36 homozygous deletion exhibit co-deletion of ENO1, in turn becoming extremely dependent on its redundant isoform ENO2 and sensitive to an overall enzymatic deficiency of enolase. Metabolomic profiling of ENO1-deleted glioma cells treated with an enolase inhibitor revealed a profound decrease in TCA cycle metabolites, which correlated with cell-line specific sensitivity to enolase inhibition, highlighting the importance of glycolysis derived pyruvate for anaplerosis. Correspondingly, the toxicity of the enolase inhibitor was significantly attenuated by exogenous supplementation of supraphysiological levels of anaplerotic substrates including pyruvate. These findings led us to hypothesize that cancer cells with ENO1 homozygous deletions treated with an enolase inhibitor might show exceptional sensitivity to inhibition of glutaminolysis because of reduced anaplerotic flow from glycolysis. We found that ENO1-deleted cells indeed exhibited selective sensitivity to the glutaminase inhibitor CB-839, and this sensitivity was also attenuated by exogenous supplementation of anaplerotic substrates including pyruvate. Despite these promising in vitro results, the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for 1p36-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of the in vitro and in vivo environments as the potential reasons for the differential efficacy of metabolism targeted therapies in in vitro and in vivo systems.\u003c/p\u003e","manuscriptTitle":"Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-15 21:24:24","doi":"10.21203/rs.3.rs-125147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-01-11T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2021-01-11T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-06T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-12-16T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-12-15T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-14T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-12-07T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-07T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-06T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-06T23:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a22c85f3-11f2-4fd3-a86d-c23a8f739316","owner":[],"postedDate":"December 15th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1440034,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-05-29T22:43:12+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-15 21:24:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-125147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-125147","identity":"rs-125147","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}