PKM2Isoform-Specific Deletion Reveals
a Differential Requirement for Pyruvate Kinase in Tumor Cells
William J.Israelsen,1Talya L.Dayton,1Shawn M.Davidson,1Brian P.Fiske,1Aaron M.Hosios,1Gary Bellinger,1Jie Li,2 Yimin Yu,1Mika Sasaki,3James W.Horner,4,13Laura N.Burga,3Jianxin Xie,5Michael J.Jurczak,6Ronald A.DePinho,4,14 Clary B.Clish,7Tyler Jacks,1Richard G.Kibbey,6,8Gerburg M.Wulf,3Dolores Di Vizio,9,10,11Gordon B.Mills,2
Lewis C.Cantley,3,12and Matthew G.Vander Heiden1,4,*
1Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology,Cambridge,MA02139,USA
2Department of Systems Biology,University of Texas M.D.Anderson Cancer Center,Houston,TX77030,USA
3Department of Medicine,Division of Signal Transduction,Beth Israel Deaconess Medical Center,Boston,MA02115,USA
4Department of Medical Oncology,Dana-Farber Cancer Institute,Boston,MA02115,USA
5Cell Signaling Technology,Danvers,MA01923,USA
6Department of Internal Medicine,Yale University School of Medicine,New Haven,CT06510,USA
7Metabolite Profiling Platform,Broad Institute,Cambridge,MA02142,USA
8Department of Cellular and Molecular Physiology,Yale University School of Medicine,New Haven,CT06510,USA
9Division of Cancer Biology and Therapeutics,Cedars-Sinai Medical Center,Los Angeles,CA90048,USA
10The Urological Diseases Research Center,Boston Children’s Hospital,Boston,MA02115,USA
11Department of Surgery,Harvard Medical School,Boston,MA02115,USA
12Department of Systems Biology,Harvard Medical School,Boston,MA02115,USA
13Present address:Institute for Applied Cancer Science and the Department of Genomic Medicine,University of Texas M.D.Anderson Cancer Center,Houston,TX77030,USA
14Present address:Department of Cancer Biology,University of Texas M.D.Anderson Cancer Center,Houston,TX77030,USA
*Correspondence:mvh@mit.edu
/10.ll.2013.09.025
SUMMARY
The pyruvate kinase M2isoform(PKM2)is ex-pressed in cancer and plays a role in regulating anabolic metabolism.To determine whether PKM2 is required for tumor formation or growth,we gener-ated mice with a conditional allele that abolishes PKM2expression without disrupting PKM1expres-sion.PKM2deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer.PKM2null tumors displayed heterogeneous PKM1expression,with PKM1found in nonprolifer-ating tumor cells and no detectable pyruvate kinase expression in proliferating cells.This suggests that PKM2is not necessary for tumor cell proliferation and implies that the inactive state of PKM2is associated with the proliferating cell population within tumors,whereas nonproliferating tumor cells require active pyruvate kinase.Consistent with thesefindings,variable PKM2expression and het-erozygous PKM2mutations are found in human tum
ors.These data suggest that regulation of PKM2activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.INTRODUCTION
Alterations in cell metabolism are a characteristic of many cancers(Cairns et al.,2011),and the metabolic program of rapidly proliferating cancer cells supports the biomass produc-tion needed to produce daughter cells(Vander Heiden et al., 2009).The M2isoform of pyruvate kinase(PKM2)is preferentially expressed in cancer,where complex regulation of its activity is important for control of cell metabolism(Chaneton and Gottlieb, 2012;Mazurek,2011).Although most studies addressing the role of PKM2in cancer metabolism have relied on analysis of cultured cells,cell culture does not recapitulate the metabolic tumor microenvironment(Vaupel et al.,1989)or the tumor cell heterogeneity that exists in vivo(Salk et al.,2010).Knockdown of PKM2in xenograft tumors has yielded contradictory results regarding the requirement for PKM2in tumor growth(Corte´s-Cros et al.,2013;Goldberg and Sharp,2012),further highlighting the need to investigate the role of PKM2in the context of spon-taneous tumors arising in situ.
Pyruvate kinase catalyzes thefinal step in glycolysis by transferring the phosphate from phosphoenolpyruvate(PEP) to ADP,thereby generating pyruvate and ATP.In mammals, pyruvate kinase is encoded by two genes that can each pro-duce two isoforms.Tissue-specific promoters drive
expression of the PKL or PKR isoforms from the PKLR gene.PKR ex-pression is exclusive to red blood cells,whereas PKL is ex-pressed primarily in the liver,with low expression in the
kidney Cell155,397–409,October10,2013ª2013Elsevier Inc.397
(Imamura and Tanaka,1972;Mazurek,2011).All other tissues studied express a product of the PKM gene,which generates either the PKM1or PKM2isoforms by including one of two mutually exclusive exons during mRNA splicing(Noguchi et al., 1986).The regulation of PKM splicing is dependent on multiple splicing factors that bind within the PKM1and PKM2exons to promote or suppress their inclusion in the mature transcript (Clower et al.,2010;David et al.,2010;Wang et al.,2012). PKM1expression is found predominantly in differentiated adult tissues with high ATP requirements,such as the heart,brain, and muscle.PKM2is expressed during development and in many adult tissues,including the spleen,lung,and all cancers and cancer cell lines studied to date(Clower et al.,2010;Ima-mura and Tanaka,1972;Mazurek,2011).
PKM1and PKM2differ by22amino acids and have distinct regulatory properties(Mazurek,2011).Whereas PKM1forms a stable,constitutively active tetramer,PKM2activity is controlled by numerous allosteric effectors and posttranslational modifica-tions that affect its tetramer stability.Binding of fructose-1,6-bisphosphate(FBP),an upstream intermediate in glycolysis, causes PKM2to adopt a stable,active conformation similar to that of PKM1(Anastasiou et al.,2012;Christofk et al.,2008b). PKM2activation by FBP can be overridden by interaction of PKM2with tyrosine-phosphorylated proteins produced in response to growth factor signaling(Christofk et al.,2008b;Var-ghese et al.,2010).PKM2activity is reduced by other posttrans-lational modifications(Anastasiou et al.,2011;Lv et al.,2011), and metabolites other than FBP can promote PKM2activation (Chaneton et al.,2012;Keller et al.,2012).These events illustrate the complex regulation of PKM2activity,and although PKM2 can exist in active or inactive states as a glycolytic enzyme,the physiological significance of these states in cells or tumors is not well understood.
It is reported that PKM2is upregulated in cancer cells and that PKM2is the isoform expressed in all tumors.This suggests that PKM2expression provides a selective advantage over other pyruvate kinase isoforms.Selection for PKM2over PKM1during xenograft tumor growth has been observed(Ch
ristofk et al., 2008a),and downregulation of PKM2enzymatic activity by phosphotyrosine growth signaling(Christofk et al.,2008a;Hito-sugi et al.,2009;Varghese et al.,2010),cellular redox state (Anastasiou et al.,2011),and lysine acetylation(Lv et al.,2011) has been associated with tumor growth and anabolic meta-bolism.Conversely,high pyruvate kinase activity due to exoge-nous PKM1expression or pharmacological activation of PKM2 can impair tumor growth and decrease levels of metabolites critical for biosynthesis in vivo(Anastasiou et al.,2012).Taken together,these studies support a model in which the ability of PKM2to be inactivated is important for cancer cell proliferation. However,this model creates a quandary:if low pyruvate kinase activity is favored by proliferating cancer cells,why is there selection for PKM2expression in cancer and not inactivation of pyruvate kinase by gene mutation,deletion,or epigenetic silencing?
One possibility is that the enzymatically inactive,nontetramer form of pyruvate kinase has an important function in cancer outside of glycolysis.Multiple nonmetabolic functions unique to PKM2have been proposed to play a vital role in cancer cell proliferation and tumor growth(Gao et al.,2012;Luo et al., 2011;Yang et al.,2011,2012a,and2012b).In all cases,these nonmetabolic functions are found only with PKM2and not with PKM1,suggesting that one or all may be driving PKM2selection in cancer.However,it remains unknown which,if any,are the critical functions promotin
g PKM2expression in tumors. Another possibility is that PKM2is selected because it can exist in states of low or high enzymatic activity to allow for meta-bolic adaptation to different physiological situations.Although reduced PKM2activity is associated with cell proliferation,it is possible that enzyme reactivation is important for nonproliferat-ing cancer cells that constitute a sizable fraction of tumor cells in vivo.If this hypothesis is correct,there must be a subpopula-tion of tumor cells in which high pyruvate kinase activity is required.Studies of PKM2in cancer to date have relied on exogenous protein overexpression or RNA interference of PKM2expression in cell culture or xenograft tumor models (Anastasiou et al.,2011;Christofk et al.,2008a;Corte´s-Cros et al.,2013;Goldberg and Sharp,2012;Luo et al.,2011;Yang et al.,2011,2012a,and2012b).Although these methods modulate PKM2expression,they impair the endogenous,cell-autonomous regulation of pyruvate kinase.
In this study,we report a conditional allele(Pkm2fl)that allows Cre-recombinase-mediated deletion of the PKM2iso-form-specific exon.Excision of Pkm exon10selectively abro-gates PKM2protein production while still allowing PKM1splicing and protein expression.Loss of PKM2in a mouse model of breast cancer accelerates tumor formation.Mammary tumors from Pkm2fl/flmice show efficient deletion,suggesting that PKM2is not absolutely required for cell proliferation or tumor growth.Only a subset of cells in Pkm2D/D tumors show compen-satory PKM1expression,and high PKM1expression
is anticor-related with cell proliferation,supporting a model in which PKM2 expression in tumors facilitates low pyruvate kinase activity needed for proliferation.Nonsense and missense mutations in PKM2occur in human tumors.These mutations include recur-rent heterozygous stop mutations in PKM exon10,consistent with the idea that tumors tolerate or favor low pyruvate kinase activity,lending further support to a model in which regulation of PKM2glycolytic activity is driving selection for this enzyme isoform in tumors.
RESULTS
Generation and Characterization of a Conditional
PKM2Allele
Pre-mRNA transcribed from the Pkm gene is spliced to produce PKM1or PKM2mRNA by inclusion of exon9or exon10,respec-tively(Figure1A).To study the role of PKM2in tumors in vivo, we generated a mouse model that allows conditional deletion of the PKM2-specific exon10.LoxP sitesflanking exon10 were introduced into the Pkm locus of mouse embryonic stem (ES)cells using homologous recombination(Figure1A and Fig-ure S1A available online).These ES cells were used to generate chimeric mice,and mice generated from germline transmission of the targeted allele were crossed wit
h mice expressing FLP recombinase to delete the neomycin resistance gene(Neo r). Breeding of these mice resulted in transmission of the PKM2
398Cell155,397–409,October10,2013ª2013Elsevier Inc.
conditional allele (Pkm2fl)in Mendelian ratios.Pkm2fl/+and Pkm2fl/flprogeny were healthy and had no overt phenotype.The presence of the Pkm2flallele in adult mice was demon-strated by Southern blot (Figures 1B,S1B,and S1C).To facilitate husbandry and analysis,we developed a PCR-based strategy for determination of Pkm2genotypes (Figure 1C).We next sought to verify that the Pkm2flallele allows disruption of PKM2expression via Cre-mediated excision of Pkm exon 10.Mice with Pkm2fland inducible Cre recombinase (Cre-ER )alleles were crossed to produce mouse embryonic fibroblasts (MEFs)for analysis.Treatment of these MEFs with 4-hydroxytamoxifen resulted in excision of Pkm exon 10in Pkm2fl/flcells (Figure 1D),
and western blot analysis showed loss of PKM2protein in the Pkm2D /D MEFs (Figure 1E).These data confirm that the Pkm2flallele allows exon 10deletion and disruption of PKM2protein production.
PKM2Loss Accelerates Tumor Formation and Promotes Liver Metastasis in a Mouse Model of Breast Cancer
To investigate the role of PKM2in tumor formation,Pkm2conditional mice were crossed to an established model of breast cancer (Xu et al.,1999).In this model,loss of the Brca1tumor suppressor in Brca1fl/flMMTV-Cre Trp53+/Àmice results in mammary tumors by $1year of age.The Pkm2flallele
should
A
B C
D
E
Figure 1.Generation and Validation of PKM2Conditional Mice
(A)The mouse Pkm locus,targeting construct,and resulting targeted,floxed,and deleted alleles.The KpnI sites used for Southern blot analysis are marked with ‘‘K,’’and the new KpnI site introduced by the targeting vector is marked with ‘‘K*.’’The locations of the 50and 30probes used for Southern blot analysis are also shown.
(B)Southern blot analysis of KpnI-digested genomic DNA using a 50probe.Digestion of the wild-type Pkm allele yields an $8.3kb fragment,and the floxed allele yields a $6.0kb fragment.
(C)PCR genotyping of genomic DNA from Pkm2+/+,Pkm2+/fl,and Pkm2fl/flmice.Genotyping primers anneal outside of the loxP sites as indicated by arrows in (A)and produce amplicons of 509bp from the Pkm2+allele and 577bp from the Pkm2flallele.
(D)PCR genotyping of Pkm2+/+Cre-ER and Pkm2fl/flCre-ER MEFs that were treated with 4-hydroxytamoxifen (TAM)or were mock treated.The Pkm2D allele produces a 195bp amplicon.
(E)Western blot analysis of PKM2protein from MEFs as specified in (D).See also Figure S1.
Cell 155,397–409,October 10,2013ª2013Elsevier Inc.399
also undergo recombination in the Cre-expressing epithelial cells that give rise to tumors in this model.Because PKM2has been implicated as playing an important role in cancer,we expected Pkm2deletion to result in fewer tumors or delayed tumor onset.Surprisingly,Pkm2fl/flmice showed accelerated tumor-associ-ated mortality when compared to their Pkm2+/+counterparts (Figure 2A).
We characterized tumors from Pkm2+/+and Pkm2fl/flmice to investigate potential factors accounting for accelerated mortality in Pkm2fl/flmice.One possibility is that PKM2loss changes the balance of proliferation and apoptosis.Staining of
tumor sections for the proliferative marker Ki-67and for the apoptosis marker cleaved caspase-3did not reveal overt differences between the genotypes (Figure S2A).However,macroscopic metastases were observed in livers from three out of five Pkm2fl/flmice compared to none of seven PKM2+/+mice evaluated for possible metastases (Figure S2B).To our knowledge,metastasis to the liver has not been reported in this mouse model of breast cancer.
To confirm that PKM2is deleted in tumors from Pkm2fl/flmice,we examined the efficiency of Cre-mediated exon 10deletion by PCR.Qualitative deletion of Pkm exon 10was observed
modulate
in
A B
C D E
F G H
Figure 2.Pkm Exon 10Deletion in Mammary Tumors Results in Accelerated Mortality and Variable Production of PKM1mRNA
(A)Kaplan-Meier survival curve comparing Pkm2+/+and Pkm2fl/flmice with Brca1fl/flMMTV-Cre Trp53+/Àalleles.
(B)PCR genotyping of the Pkm2allele in Pkm2+/+and Pkm2D /D mammary tumors.Analysis of tail DNA from Pkm2+/+and Pkm2fl/flmice is shown as a control.(C)PKM2mRNA levels in Pkm2+/+and Pkm2D /D tumors by quantitative RT-PCR,with normal mouse tissue controls.M,muscle;H,heart;B,brain;K,kidney.(D)PKM1mRNA levels in Pkm2+/+and Pkm2D /D tumors by quantitative RT-PCR,with normal mouse tissue controls.K,kidney;M,muscle;H,heart;B,brain.(E)PKLR mRNA levels in Pkm2+/+and Pkm2D /D tumors by quantitative RT-PCR,with normal mouse tissue controls.L,liver;K,kidney.
(F)Autoradiograph of uncut (U)and PstI (P)digested Pkm cDNA amplicons.Uncut PKM1and PKM2amplicons are of identical length (band 1).PstI digests only the PKM2amplicon to produce bands 2and 3.Results from three representative Pkm2+/+tumors are shown with quantification.See Figures S2C and S2E for a schematic of how each band is generated.
(G)Autoradiograph of uncut (U)and digested PKM cDNA amplicons from a representative Pkm2D /D tumor.Uncut PKM1and PKM2amplicons are of identical length (band 1),and the amplicon corresponding to the PKM-skip mis-spliced product is marked with an arrowhead (band 3).NcoI (N)digests the PKM1am-plicon,and PstI (P)digests the PKM2amplicon.Figures S2C,S2D,and S2F show how each band is generated.(H)Quantification of PKM splicing in Pkm2D /D tumors,as determined from autoradiographs as in (G).Data are displayed as means ±SEM.n =4.See also Figure S2.
400Cell 155,397–409,October 10,2013ª2013Elsevier Inc.
all tumors derived from Pkm2fl/flmice(Figure2B),and quantita-tive PCR(qPCR)analysis of tumor transcripts showed very low levels of PKM2mRNA compared to wild-type tumors(Figure2C). Because Pkm2D/D tumor cells can potentially generate PKM1 protein by inclusion of exon9during mRNA splicing,we quanti-fied PKM1mRNA levels by qPCR.Some Pkm2D/D tumors had low PKM1expression at levels similar to Pkm2+/+tumors, whereas others had PKM1mRNA levels approaching those found in the brain,a tissue that normally expresses PKM1(Fig-ure2D).To determine whether Pkm2D/D tumors had compensa-tory expression of the other two pyruvate kinase isoforms,PKL or PKR,we examined tumor PKLR mRNA levels by qPCR.We did not observe any mR
NA expression from the Pklr gene in tumors of any genotype,suggesting that protein derived from the Pkm gene was the only pyruvate kinase present(Figure2E). PKM2Deletion Results in Production of Either PKM1
or a Mis-Spliced mRNA
We further characterized PKM mRNA splicing in tumors from Pkm2+/+and Pkm2fl/flmice.Exons9and10are of identical length but contain unique restriction enzyme sites that allow for relative quantification of PKM1and PKM2transcript levels (Clower et al.,2010;David et al.,2010).The alternatively spliced region between PKM exons8and11was amplified from Pkm2+/+and Pkm2D/D tumor cDNA and was digested for analysis (Figures S2C and S2E).Consistent with the qPCR results,PKM2 was the dominant transcript found in Pkm2+/+tumors(Figure2F). Pkm2D/D tumors displayed an overall decrease in PKM2mRNA, with PKM1message comprising a larger fraction of the total Pkm-derived mRNAs(Figure2G);however,this analysis also revealed an additional PKM splice variant not found in wild-type tumors(compare left lanes of Figures2F and2G).This mRNA species accounted for$40%of the PKM transcript in Pkm2D/D tumors(Figure2H),was identified by sequencing to result from the splicing of exon8to exon11(Figures S2D, S2F,S2G),and is hereafter referred to as PKM-skip.
Splicing of PKM pre-mRNA to produce PKM2transcript involves repression of exon9inclusion and activation of exon 10inclusion by the binding of specific splicing factors to se-quences within the alternatively spliced exons(Clower et al., 2010;David et al.,2010;Wang et al.,2012).Thus,the absence of exon10together with repression of exon9likely explains the presence of the PKM-skip mRNA species in Pkm2D/D tumors (Chen et al.,2012).Splicing of PKM exon8to11causes a frame-shift at the exon8–11junction,which results in38missense codons and a premature stop codon>500base pairs upstream of the exon11–12junction(Figure S2G).Aberrant splicing of PKM transcript to create a premature stop codon upstream of the exon11–12junction leads to message degradation by nonsense-mediated decay(Chen et al.,2012).
PKM2Deletion Results in Variable PKM1Protein
Levels but No PKM-skip Protein
Because deletion of Pkm2exon10does not always lead to exon9inclusion and production of PKM1mRNA and because PKM1mRNA levels varied widely across tumors,we determined the effects of Pkm2deletion on pyruvate kinase protein levels in Pkm2D/D tumors.Western blotting of tumor lysates confirmed the loss of PKM2protein in Pkm2D/D tumors(Figure3A).Variable amounts of PKM1
protein were observed in Pkm2D/D tumor ly-sates,and overall expression of pyruvate kinase was decreased in Pkm2D/D tumors relative to Pkm2+/+tumors,such that even tumors with the highest PKM1expression had lower levels than PKM1-expressing skeletal muscle.
We next determined whether the PKM-skip mRNA generates a protein product in Pkm2D/D tumors.Wild-type PKM1and PKM2are both531residues long with subunit masses of$58 kD,whereas the predicted PKM-skip protein is418residues long with a predicted mass of45kD(Figure S3A).To verify that we could detect PKM-skip protein via western blot,PKM-skip cDNA was expressed li with an N-terminal63-His tag and was purified using affinity chromatography.The same approach is used to produce PKM2protein that is active both as a glycolytic enzyme(Anastasiou et al.,2012;Dombrauckas et al.,2005)and as a putative protein kinase(Gao et al.,2012; Yang et al.,2012a).Some PKM-skip product was recovered from the soluble fraction after bacterial lysis;however,the ex-pected protein was accompanied by a series of smaller pro-ducts,consistent with partial degradation of an unstable protein li(Figure S3B).Both the full-length and smaller polypep-tides were recognized by two different anti-PKM antibodies during western blot analysis(Figure S3C).We next performed western blot analysis of tumor lysates using recombinant PKM-skip protein as a control;however,we failed to detect a band smaller than the full-length protein i
n any tumors(Fig-ure3B).These data show that PKM-skip protein is not present in appreciable quantities in Pkm2D/D tumors.
Because small amounts of catalytic activity are potentially meaningful,we considered that levels of putative PKM-skip below our detection limit might retain some PKM2activity.By comparison with published PKM2protein structures(Christofk et al.,2008b;Dombrauckas et al.,2005),PKM-skip protein is predicted to lack the FBP binding site and the dimer-dimer inter-face found in the native PKM2tetramer(Figure S3D).However, with a few amino acid changes,the predicted PKM-skip mono-mer retains the primary structure necessary to produce the domains hosting the active site(Figure S3E).We thus evaluated whether recombinant PKM-skip retained catalytic activity and whether this protein could form dimers or tetramers.Using size exclusion chromatography,we found that recombinant PKM-skip eluted at volumes consistent with it being a population of 45kD or smaller monomers(Figure S3F).PKM-skip protein was>230,000-fold less active per microgram than recombinant PKM2for glycolytic pyruvate kinase activity(Figure S3G)and had no detectable protein kinase activity(Figure S3H),suggest-ing that PKM-skip protein would not have meaningful catalytic activity even if it were present in Pkm2D/D tumors.Taken together,these data suggest that PKM-skip protein is not a confounding factor in the analysis of Pkm2D/D tumors and that PKM exon10deletion effectively precludes PKM2protein pro-duction while still allowing cells to express PKM1.
Expression of PKM1in PKM2Knockout Tumors
Is Spatially Heterogeneous
To understand which cells in Pkm2D/D tumors express PKM1, we performed immunohistochemistry(IHC)onfixed tumor Cell155,397–409,October10,2013ª2013Elsevier Inc.401
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