{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:57:49Z","timestamp":1785430669228,"version":"3.56.0"},"reference-count":48,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003407","name":"Ministry of Education, Universities and Research","doi-asserted-by":"publisher","award":["PRIN 393 2017NTK4HY_002"],"award-info":[{"award-number":["PRIN 393 2017NTK4HY_002"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003407","name":"Ministry of Education, Universities and Research","doi-asserted-by":"publisher","award":["PROGETTO PNRR THE\u2014TUSCANY HEALTH ECOSYSTEM\u2014SPOKE N. 7, CUP B63C22000680007"],"award-info":[{"award-number":["PROGETTO PNRR THE\u2014TUSCANY HEALTH ECOSYSTEM\u2014SPOKE N. 7, CUP B63C22000680007"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003407","name":"Ministry of Education, Universities and Research","doi-asserted-by":"publisher","award":["C.N.3 - SPOKE N.5, CUP B63C2200061 0006"],"award-info":[{"award-number":["C.N.3 - SPOKE N.5, CUP B63C2200061 0006"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]},{"award":["PRIN 393 2017NTK4HY_002"],"award-info":[{"award-number":["PRIN 393 2017NTK4HY_002"]}],"id":[{"id":"https:\/\/ror.org\/0166hxq48","id-type":"ROR","asserted-by":"publisher"}]},{"award":["PROGETTO PNRR THE\u2014TUSCANY HEALTH ECOSYSTEM\u2014SPOKE N. 7, CUP B63C22000680007"],"award-info":[{"award-number":["PROGETTO PNRR THE\u2014TUSCANY HEALTH ECOSYSTEM\u2014SPOKE N. 7, CUP B63C22000680007"]}],"id":[{"id":"https:\/\/ror.org\/0166hxq48","id-type":"ROR","asserted-by":"publisher"}]},{"award":["C.N.3 - SPOKE N.5, CUP B63C2200061 0006"],"award-info":[{"award-number":["C.N.3 - SPOKE N.5, CUP B63C2200061 0006"]}],"id":[{"id":"https:\/\/ror.org\/0166hxq48","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Hedgehog (Hh) signaling regulates cell survival and microenvironmental responses in various cancers, but its role in metabolic adaptation to the hypoxic bone marrow microenvironment in myeloid malignancies remains insufficiently defined. K562, KU812, and U937 cells were cultured under normoxic or hypoxic conditions and treated with the Smoothened (Smo) antagonist Cyclopamine or the agonist SAG (Smoothened Agonist). Cell proliferation, apoptosis- and autophagy-related markers, glycolysis-associated proteins, and metabolic parameters were assessed by viability assays, Western blotting, immunofluorescence, and biochemical assays. Selected findings were further evaluated in CRISPR\/Cas9-mediated Smo knockout cells. Smo inhibition reduced cell proliferation, increased PARP cleavage, and decreased BNIP3 expression under both oxygen conditions. It also downregulated glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase (LDH), monocarboxylate transporter 1 (MCT1), carbonic anhydrases IX and XII (CAIX and CAXII), and was associated with reduced glucose consumption, lactate production, and ATP levels. These changes were associated with modulation of the AMPK\u2013mTOR axis and with reduced phosphorylation of mTOR downstream effectors. These findings support a role for Hh signaling in hypoxia-driven metabolic adaptation in myeloid malignancy cell models and suggest a functional link between Smo activity, glycolytic remodeling, and AMPK-mTOR-related signaling.<\/jats:p>","DOI":"10.3390\/ijms27146324","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T03:12:38Z","timestamp":1784257958000},"page":"6324","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hedgehog Signaling Regulates Hypoxia-Associated Metabolic Adaptation in Myeloid Leukemia In Vitro Cell Models"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7712-1474","authenticated-orcid":false,"given":"Irene","family":"Filippi","sequence":"first","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Monaci","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlo","family":"Aldinucci","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-2636-6443","authenticated-orcid":false,"given":"Alessandro","family":"Falsini","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-8223-3757","authenticated-orcid":false,"given":"Massimo","family":"Bardotti","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federica","family":"Coppola","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9600-2486","authenticated-orcid":false,"given":"Antonella","family":"Naldini","sequence":"additional","affiliation":[{"name":"Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0771-042X","authenticated-orcid":false,"given":"Fabio","family":"Carraro","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1039\/C5NP00153F","article-title":"I only have eye for ewe: The discovery of cyclopamine and development of Hedgehog pathway-targeting drugs","volume":"33","author":"Chen","year":"2016","journal-title":"Nat. Prod. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1559","DOI":"10.1126\/science.1073733","article-title":"Medulloblastoma growth inhibition by hedgehog pathway blockade","volume":"297","author":"Berman","year":"2002","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2383","DOI":"10.1080\/10428190802510315","article-title":"The hedgehog inhibitor cyclopamine induces apoptosis in leukemic cells in vitro","volume":"49","author":"Warzecha","year":"2008","journal-title":"Leuk. Lymphoma"},{"key":"ref_4","first-page":"4629","article-title":"Cyclopamine and quercetin suppress the growth of leukemia and lymphoma cells","volume":"29","author":"Kawahara","year":"2009","journal-title":"Anticancer Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1016\/S0006-3495(01)75733-5","article-title":"Modeling pO2 distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models","volume":"81","author":"Chow","year":"2001","journal-title":"Biophys. J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5431","DOI":"10.1073\/pnas.0701152104","article-title":"Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia","volume":"104","author":"Parmar","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","first-page":"375","article-title":"Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: Serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches","volume":"116","author":"Winkler","year":"2010","journal-title":"Blood J. Am. Soc. Hematol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.stem.2010.06.020","article-title":"Regulation of the HIF-1\u03b1 level is essential for hematopoietic stem cells","volume":"7","author":"Takubo","year":"2010","journal-title":"Cell Stem Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.stem.2012.10.011","article-title":"Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells","volume":"12","author":"Takubo","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Giuntini, G., Coppola, F., Falsini, A., Filippi, I., Monaci, S., Naldini, A., and Carraro, F. (2022). Role of the hedgehog pathway and CAXII in controlling melanoma cell migration and invasion in hypoxia. Cancers, 14.","DOI":"10.3390\/cancers14194776"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Giuntini, G., Monaci, S., Cau, Y., Mori, M., Naldini, A., and Carraro, F. (2020). Inhibition of melanoma cell migration and invasion targeting the hypoxic tumor associated CAXII. Cancers, 12.","DOI":"10.3390\/cancers12103018"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Falsini, A., Giuntini, G., Mori, M., Ghirga, F., Quaglio, D., Cucinotta, A., Coppola, F., Filippi, I., Naldini, A., and Botta, B. (2024). Hedgehog Pathway Inhibition by Novel Small Molecules Impairs Melanoma Cell Migration and Invasion under Hypoxia. Pharmaceuticals, 17.","DOI":"10.3390\/ph17020227"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9799","DOI":"10.1002\/jcp.26947","article-title":"Inhibition of smoothened in breast cancer cells reduces CAXII expression and cell migration","volume":"233","author":"Guerrini","year":"2018","journal-title":"J. Cell. Physiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1016\/j.bbrc.2019.06.040","article-title":"Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancer cells","volume":"516","author":"Guerrini","year":"2019","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"11039","DOI":"10.1111\/jcmm.17027","article-title":"Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions","volume":"25","author":"Chen","year":"2021","journal-title":"J. Cell. Mol. Med."},{"key":"ref_16","first-page":"2595","article-title":"HIF1\u03b1 is required for survival maintenance of chronic myeloid leukemia stem cells","volume":"119","author":"Zhang","year":"2012","journal-title":"Blood J. Am. Soc. Hematol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1038\/s41598-018-19182-8","article-title":"Role of hypoxia in Diffuse Large B-cell Lymphoma: Metabolic repression and selective translation of HK2 facilitates development of DLBCL","volume":"8","author":"Bhalla","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1007\/s00277-003-0650-0","article-title":"Prognosis of older patients with acute myeloid leukemia receiving either induction or noncurative treatment: A single-center retrospective study","volume":"82","author":"Behringer","year":"2003","journal-title":"Ann. Hematol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"931","DOI":"10.7326\/0003-4819-115-12-931","article-title":"High serum lactate dehydrogenase level as a marker for drug resistance and short survival in multiple myeloma","volume":"115","author":"Dimopoulos","year":"1991","journal-title":"Ann. Intern. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1126\/science.1160809","article-title":"Understanding the Warburg effect: The metabolic requirements of cell proliferation","volume":"324","author":"Cantley","year":"2009","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1393","DOI":"10.1111\/j.1742-4658.2007.05686.x","article-title":"Energy metabolism in tumor cells","volume":"274","author":"Saavedra","year":"2007","journal-title":"FEBS J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1158\/0008-5472.CAN-09-3537","article-title":"Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62\/SQSTM1 expression and AMPK activation","volume":"70","author":"Puissant","year":"2010","journal-title":"Cancer Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2715","DOI":"10.2174\/092986711796011201","article-title":"Targeting the phosphatidylinositol 3-kinase\/Akt\/mammalian target of rapamycin signaling network in cancer stem cells","volume":"18","author":"Martelli","year":"2011","journal-title":"Curr. Med. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1895","DOI":"10.1101\/gad.17420111","article-title":"AMP-activated protein kinase\u2014An energy sensor that regulates all aspects of cell function","volume":"25","author":"Hardie","year":"2011","journal-title":"Genes. Dev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1309\/AJCPPPZ1WFVGNE4A","article-title":"Molecular pathology of myeloproliferative neoplasms","volume":"133","author":"Klco","year":"2010","journal-title":"Am. J. Clin. Pathol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1007\/s00204-014-1433-1","article-title":"The Hedgehog pathway: Role in cell differentiation, polarity and proliferation","volume":"89","author":"Jia","year":"2015","journal-title":"Arch. Toxicol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1038\/nature13034","article-title":"Direct measurement of local oxygen concentration in the bone marrow of live animals","volume":"508","author":"Spencer","year":"2014","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1016\/S0960-9822(01)00178-6","article-title":"The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking","volume":"11","author":"Carrillo","year":"2001","journal-title":"Curr. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.cmet.2005.11.012","article-title":"Hedgehog signaling plays a conserved role in inhibiting fat formation","volume":"3","author":"Suh","year":"2006","journal-title":"Cell Metab."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1634\/stemcells.2007-0974","article-title":"Hedgehog signaling alters adipocyte maturation of human mesenchymal stem cells","volume":"26","author":"Fontaine","year":"2008","journal-title":"Stem Cells"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e13308","DOI":"10.7554\/eLife.13308","article-title":"Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis","volume":"5","author":"Rennert","year":"2016","journal-title":"eLife"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"105825","DOI":"10.1016\/j.jsbmb.2021.105825","article-title":"Relationship between lipid metabolism and Hedgehog signaling pathway","volume":"209","author":"Gu","year":"2021","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"29083","DOI":"10.1074\/jbc.270.49.29083","article-title":"Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences","volume":"270","author":"Ebert","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4015","DOI":"10.2741\/3509","article-title":"Mitochondria: From bioenergetics to the metabolic regulation of carcinogenesis","volume":"14","author":"Nadege","year":"2009","journal-title":"Front. Biosci."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Mason, S. (2017). Lactate shuttles in neuroenergetics\u2014Homeostasis, allostasis and beyond. Front. Neurosci., 11.","DOI":"10.3389\/fnins.2017.00043"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.tcb.2015.10.013","article-title":"AMPK: An energy-sensing pathway with multiple inputs and outputs","volume":"26","author":"Hardie","year":"2016","journal-title":"Trends Cell Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41580-022-00547-x","article-title":"New insights into activation and function of the AMPK","volume":"24","author":"Steinberg","year":"2023","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1016\/j.cell.2012.09.021","article-title":"Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat","volume":"151","author":"Teperino","year":"2012","journal-title":"Cell"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.bbcan.2017.06.003","article-title":"New paradigms for the Hedgehog signaling network in mammary gland development and breast Cancer","volume":"1868","author":"Monkkonen","year":"2017","journal-title":"Biochim. Biophys. Acta BBA Rev. Cancer"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1016\/j.exphem.2012.01.003","article-title":"Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through \u03b2-catenin signaling","volume":"40","author":"Su","year":"2012","journal-title":"Exp. Hematol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3012","DOI":"10.1182\/blood-2006-04-019109","article-title":"Transfer of differentiation signal by membrane microvesicles harboring hedgehog morphogens","volume":"108","author":"Martinez","year":"2006","journal-title":"Blood"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1016\/j.leukres.2012.02.012","article-title":"Gli inhibitor GANT61 causes apoptosis in myeloid leukemia cells and acts in synergy with rapamycin","volume":"36","author":"Pan","year":"2012","journal-title":"Leuk. Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Holzwarth, C., Vaegler, M., Gieseke, F., Pfister, S.M., Handgretinger, R., Kerst, G., and M\u00fcller, I. (2010). Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells. BMC Cell Biol., 11.","DOI":"10.1186\/1471-2121-11-11"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1111\/j.1474-9726.2007.00336.x","article-title":"Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan","volume":"6","author":"Fehrer","year":"2007","journal-title":"Aging Cell"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"a008771","DOI":"10.1101\/cshperspect.a008771","article-title":"mTOR-dependent cell survival mechanisms","volume":"4","author":"Hung","year":"2012","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.molcel.2008.03.003","article-title":"AMPK phosphorylation of raptor mediates a metabolic checkpoint","volume":"30","author":"Gwinn","year":"2008","journal-title":"Mol. Cell"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Monaci, S., Aldinucci, C., Rossi, D., Giuntini, G., Filippi, I., Ulivieri, C., Marotta, G., Sozzani, S., Carraro, F., and Naldini, A. (2020). Hypoxia Shapes Autophagy in LPS-Activated Dendritic Cells. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.573646"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"4282","DOI":"10.1002\/jcp.26247","article-title":"The Shc protein RAI promotes an adaptive cell survival program in hypoxic neuroblastoma cells","volume":"233","author":"Criscuoli","year":"2018","journal-title":"J. Cell Physiol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6324\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,18]],"date-time":"2026-07-18T04:29:53Z","timestamp":1784348993000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6324"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,16]]},"references-count":48,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ijms27146324"],"URL":"https:\/\/doi.org\/10.3390\/ijms27146324","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,16]]}}}