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Pyruvate Kinase Inhibits Proliferation During Postnatal Cerebellar Neurogenesis and Suppresses Medulloblastoma Formation Katherine Tech1,2, Andrey P

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Pyruvate Kinase Inhibits Proliferation During Postnatal Cerebellar Neurogenesis and Suppresses Medulloblastoma Formation Katherine Tech1,2, Andrey P Published OnlineFirst May 17, 2017; DOI: 10.1158/0008-5472.CAN-16-3304 Cancer Molecular and Cellular Pathobiology Research Pyruvate Kinase Inhibits Proliferation during Postnatal Cerebellar Neurogenesis and Suppresses Medulloblastoma Formation Katherine Tech1,2, Andrey P. Tikunov1, Hamza Farooq3,4, A. Sorana Morrissy3,4, Jessica Meidinger2, Taylor Fish2, Sarah C. Green1, Hedi Liu2, Yisu Li5, Andrew J. Mungall5, Richard A. Moore5, Yussanne Ma5, Steven J.M. Jones5, Marco A. Marra5, Matthew G. Vander Heiden6,7, Michael D. Taylor3,4,8,9, Jeffrey M. Macdonald1, and Timothy R. Gershon2,10,11 Abstract Aerobic glycolysis supports proliferation through unresolved sively expressed the less active PKM2. Isoform-specific Pkm2 mechanisms. We have previously shown that aerobic glycolysis is deletion in CGNPs blocked all Pkm expression. Pkm2-deleted required for the regulated proliferation of cerebellar granule CGNPs showed reduced lactate production and increased neuron progenitors (CGNP) and for the growth of CGNP-derived SHH-driven proliferation. 13C-flux analysis showed that Pkm2 medulloblastoma. Blocking the initiation of glycolysis via dele- deletion reduced the flow of glucose carbons into lactate and tion of hexokinase-2 (Hk2) disrupts CGNP proliferation and glutamate without markedly increasing glucose-to-ribose flux. restricts medulloblastoma growth. Here, we assessed whether Pkm2 deletion accelerated tumor formation in medulloblasto- disrupting pyruvate kinase-M (Pkm), an enzyme that acts in the ma-prone ND2:SmoA1 mice, indicating the disrupting PKM terminal steps of glycolysis, would alter CGNP metabolism, releases CGNPs from a tumor-suppressive effect. These findings proliferation, and tumorigenesis. We observed a dichotomous show that distal and proximal disruptions of glycolysis have pattern of PKM expression, in which postmitotic neurons opposite effects on proliferation, and that efforts to block the throughout the brain expressed the constitutively active PKM1 oncogenic effect of aerobic glycolysis must target reactions isoform, while neural progenitors and medulloblastomas exclu- upstream of PKM. Cancer Res; 77(12); 1–14. Ó2017 AACR. Introduction amplifying cells that proliferate in the postnatal brain (3, 4) that utilize aerobic glycolysis during normal brain development (5). Increased aerobic glycolysis, a common feature of proliferating CGNPs are also cells-of-origin for SHH-subgroup medulloblas- cells during developmental and malignant growth (1, 2), has been toma (6, 7). Medulloblastomas coopt developmentally regulated proposed as a target for anticancer therapy. CGNPs are transit programs of CGNPs (6, 7), including the metabolic phenotype of increased aerobic glycolysis (5, 8). Understanding how aerobic 1 glycolysis supports CGNP proliferation and medulloblastoma Joint Department of Biomedical Engineering, University of North Carolina at tumorigenesis is essential for designing metabolically directed Chapel Hill and North Carolina State University, Chapel Hill, North Carolina. 2Department of Neurology, University of North Carolina School of Medicine, medulloblastoma therapies. Chapel Hill, North Carolina. 3Developmental & Stem Cell Biology Program, The During postnatal brain development, CGNPs proliferate in Hospital for Sick Children, Toronto, Ontario, Canada. 4The Arthur and Sonia the cerebellum in response to locally secreted Sonic Hedgehog Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, (SHH), generating the largest neuron population in the mam- Ontario, Canada. 5Michael Smith Genome Sciences Centre, BC Cancer Agency, – 6 malian brain (9 11). In synchrony with increased CGNP pro- Vancouver, British Columbia, Canada. David H. Koch Institute for Integrative liferation, SHH signaling induces hexokinase-2 (HK2) and Cancer Research, Massachusetts Institute of Technology, Cambridge, Massa- chusetts. 7Dana-Farber Cancer Institute, Boston, Massachusetts. 8Department of aerobic glycolysis (5, 8). Approximately 30% of medulloblas- Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, toma patients show SHH pathway activation (12, 13) and mice Canada. 9Division of Neurosurgery, The Hospital for Sick Children, Toronto, with activating SHH pathway mutations develop spontaneous Ontario, Canada. 10Lineberger Comprehensive Cancer Center, University of medulloblastomas that recapitulate the human disease 11 North Carolina School of Medicine, Chapel Hill, North Carolina. UNC Neurosci- (14, 15). These tumors arise from CGNPs and, like their ence Center, University of North Carolina School of Medicine, Chapel Hill, North cells-of-origin, upregulate HK2 and aerobic glycolysis (5, 8). Carolina. CGNP proliferation and SHH-driven tumorigenesis in mice Note: Supplementary data for this article are available at Cancer Research typify aerobic glycolysis supporting tumorigenesis arising from Online (http://cancerres.aacrjournals.org/). developmental growth. Corresponding Author: Timothy R. Gershon, Department of Neurology, Uni- We have previously shown that conditional deletion of Hk2 in versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: 919-966- the developing brain blocks SHH-induced aerobic glycolysis, 3618; Fax: 919-966-2922; E-mail: [email protected] disrupts CGNP differentiation, and reduces medulloblastoma doi: 10.1158/0008-5472.CAN-16-3304 growth, extending the survival of medulloblastoma-prone mice Ó2017 American Association for Cancer Research. (5). These findings demonstrate the importance of aerobic www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst May 17, 2017; DOI: 10.1158/0008-5472.CAN-16-3304 Tech et al. glycolysis in development and cancer, suggesting that blocking Materials and Methods glycolysis through HK2 inhibition may produce a clinically sig- Animals nificant antitumor effect. Development of selective HK2 inhibi- All wild-type and genetically engineered mice were main- tors for anticancer therapy, however, has been problematic (16). tained on the C57/Bl6 background with at least 5 backcrosses. Whether targeting glycolysis downstream of HK2 would similarly ND2:SmoA1 mice were provided by Dr. James Olson (Fred inhibit tumor growth is unknown. To determine whether the Hutchinson Cancer Research Center, Seattle, WA). Math1-Cre distal portion of the glycolytic pathway is required for the pro- mice were provided by Dr. David Rowitch (University of liferation of normal and transformed CGNPs, we analyzed the California San Francisco, San Francisco, CA) and Robert Wechs- effect of disrupting pyruvate kinase (PK) during cerebellar neu- ler-Reya (Sanford-Burnham Medical Research Institute, La Jolla, rogenesis and medulloblastoma formation. CA). Eva Anton (University of North Carolina at Chapel Hill, While HK2 initiates glycolysis, PK catalyzes the final step, fl/fl Chapel Hill, NC) provided hGFAP-Cre mice. Pkm2 mice were converting phosphoenolpyruvate (PEP) and ADP into pyruvate generously shared by Dr. Matthew Vander Heiden (Massachu- and ATP (17). Pkm is the pyruvate kinase expressed in the brain setts Institute of Technology, Cambridge, MA). Medulloblas- (18). Alternative splicing of a single exon from Pkm gives rise to toma-prone mice were monitored daily for abnormalities of either Pkm1, which includes exon 9, or Pkm2, where exon 9 head shape and movement. At the onset of tumor symptoms, replaces exon 10 (19). PKM1 is constitutively active and typically such as weight loss, ataxia, and impaired movement, animals expressed in differentiated cells, while PKM2 has a regulated were sacrificedandsurvivaltimetoonsetofsymptomswas activity and is commonly expressed in cancer (20–24). PKM2 considered the event-free survival. can be activated by fructose-1,6-bisphosphate, an upstream gly- For 5-ethynyl-20-deoxyuridine (EdU) experiments, mice were colytic intermediate (23, 24) and inactivated by interaction with intraperitoneally injected with EdU (#A10044, Life Technologies) tyrosine-phosphorylated proteins responding to growth factor at 40 mg/kg in 50 mL of HBSS and dissected 24 hours later. Brains receptor signaling (20, 21, 25). While the regulated activity of were fixed in 4% paraformaldehyde in 1Â PBS for 24 hours at 4C, PKM2 may allow proliferating cells to adjust their metabolism to then processed for histology. All animal handling and protocols dynamic requirements (2), the specific benefit of low PKM2 were carried out in accordance with established NIH practices activity has not been identified. and approved under University of North Carolina Institutional PKM2 is expressed in diverse cancers, suggesting a role in cancer Animal Care and Use Committee #13-121.0 and 15-306.0. growth. Xenograft tumors engineered to overexpress either iso- form showed that PKM2 expression confers a relative growth Cell culture techniques advantage (26). Other studies have reported nonmetabolic, CGNPs were isolated and cultured as previously described growth-promoting functions such as transcriptional regulation (5, 34). Briefly, cerebella were dissected from P5 pups, disso- (27, 28) and histone phosphorylation (29, 30). However, iso- ciated,andallowedtoadheretocoatedculturewellsin fi form-speci c deletion studies show that PKM2 is not required for DMEM/F12 (#11320, Life Technologies) with 25 mmol/L KCl, fi Pkm2 tumor formation. Aged mice with isoform-speci c deletion supplemented with FBS and N2. After 4 hours, media were develop liver cancer (31) and breast
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