PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth

Article

PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth Graphical Abstract

Authors Mami Morita, Taku Sato, Miyuki Nomura, ..., Hiroshi Shima, Makoto Maemondo, Nobuhiro Tanuma

Correspondence [email protected]

In Brief The relative importance of PKM isoforms in tumor growth has been controversial. Morita et al. show that PKM1 promotes the growth of multiple tumor models using mouse lines expressing PKM1 or PKM2 from the endogenous Pkm locus. PKM1 is expressed in human SCLC, and it is important for SCLC cell proliferation.

Highlights d

PKM1 promotes tumor growth cell intrinsically in some contexts

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PKM1 activates glucose catabolism without interfering with biosynthetic pathways

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PKM1-dependent autophagy/mitophagy contributes to malignancy

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Expression of PKM1, but not PKM2, is sufficient to support SCLC cell proliferation

Morita et al., 2018, Cancer Cell 33, 355–367 March 12, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.ccell.2018.02.004

Cancer Cell

Article PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth Mami Morita,1,6,13,15 Taku Sato,1,7,15 Miyuki Nomura,1,15 Yoshimi Sakamoto,1 Yui Inoue,1 Ryota Tanaka,1,7 Shigemi Ito,1 Koreyuki Kurosawa,1 Kazunori Yamaguchi,2 Yuki Sugiura,4 Hiroshi Takizaki,5 Yoji Yamashita,1 Ryuichi Katakura,1 Ikuro Sato,3 Masaaki Kawai,1 Yoshinori Okada,7 Hitomi Watanabe,9 Gen Kondoh,9 Shoko Matsumoto,10 Ayako Kishimoto,10 Miki Obata,11 Masaki Matsumoto,12 Tatsuro Fukuhara,13 Hozumi Motohashi,8 Makoto Suematsu,4 Masaaki Komatsu,11 Keiichi I. Nakayama,12 Toshio Watanabe,10 Tomoyoshi Soga,14 Hiroshi Shima,1,5 Makoto Maemondo,6,13 and Nobuhiro Tanuma1,5,16,* 1Division

of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan of Molecular and Cellular Oncology, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan 3Tissue Bank, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan 4Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan 5Division of Cancer Molecular Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 6Division of Respiratory Oncology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 7Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan 8Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan 9Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan 10Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women’s University, Nara 630-8506, Japan 11Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan 12Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyusyu University, Fukuoka 812-8582, Japan 13Department of Respiratory Medicine, Miyagi Cancer Center Hospital, Natori 981-1293, Japan 14Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0052, Japan 15These authors contributed equally 16Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.ccell.2018.02.004 2Division

SUMMARY

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including smallcell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

Significance PKM isoform switching has been of interest since the discovery that PKM2 is highly expressed in cancer cells and promotes biosynthetic metabolism and the Warburg effect. One conclusion has been that PKM2 is essential for tumor growth. However, mice deficient of PKM2 specifically have been reported to show enhanced tumorigenesis. We show that PKM1, rather than PKM2, exhibits tumor-promoting function cell autonomously in a KrasG12D mouse model and in KRASG12V mouse embryo fibroblasts, and that pulmonary NETs, including SCLC, express PKM1 required for cell proliferation in that context. These findings challenge the idea that limiting glucose catabolism by PKM2 is a prerequisite for tumorigenesis, and provide a rationale for targeting PKM1 therapeutically in SCLC, in which few druggable mutations have been identified. Cancer Cell 33, 355–367, March 12, 2018 ª 2018 Elsevier Inc. 355

INTRODUCTION Limited glucose oxidation, even in the presence of sufficient oxygen, is known as the Warburg effect. Many reports suggest that metabolic phenotypes resembling the Warburg effect are cancer abetting in tumor cells (Christofk et al., 2008; Vander Heiden et al., 2009; Vander Heiden and DeBerardinis, 2017), although recent studies present conflicting findings. For example, glucose oxidation is activated in a malignant subset of human melanomas (Vazquez et al., 2013), in a highly metastatic population of mammary tumors (LeBleu et al., 2014), and in lung tumors in vivo in patients (Hensley et al., 2016). It is also recently reported that oncogenic Kras activates oxidative glucose metabolism as well as fermentation (Kerr et al., 2016). Activity of the glycolytic enzyme pyruvate kinase M (PKM) reportedly determines the fate of glucose-derived carbon (Christofk et al., 2008; Dayton et al., 2016b). PKM gives rise to splice variants encoding PKM1 and PKM2 isoforms. Both convert phosphoenolpyruvate (PEP) to pyruvate in glycolysis. Unlike constitutively active PKM1, PKM2 is activated only when cellular levels of allosteric activator(s) increase (Chaneton et al., 2012; Keller et al., 2012). The conditionally activated property of PKM2 activity maintains glycolysis flux at a lower rate and limits glucose oxidation (Jiang and Deberardinis, 2012). Most cancer cells predominantly express PKM2 over PKM1, an activity implicated in the Warburg effect and in metabolic rewiring in which glucose-derived carbons are diverted from catabolic to biosynthetic pathways (Chaneton and Gottlieb, 2012; Dayton et al., 2016b; Ward and Thompson, 2012). The mechanisms by which PKM2’s lower activity is associated with elevated lactate formation in tumors are unknown. PKM2 expression reportedly confers metabolic advantages to some cell lines grown under hypoxia (Christofk et al., 2008). However, mice deficient of PKM2 specifically (PKM2-knockout [KO]) exhibit enhanced tumorigenesis in several experimental models (Dayton et al., 2016a; Israelsen et al., 2013; Tech et al., 2017). As such, it remains unclear whether PKM2 is cancer promoting or suppressing. Here, we address this issue. RESULTS Knockin Mice Express Specific PKM Isoforms PKM2-KO mice, which show PKM2 loss due to deletion of a PKM2-specific exon in Pkm, display compensatory and partial expression of PKM1 (Dayton et al., 2016a; Israelsen et al., 2013; Lunt et al., 2015). However, most transcripts from the mutant allele are cryptically spliced, and only low levels of functional PKM1 mRNA are present, hampering analysis of PKM1 or PKM2 function in this model. To circumvent these problems, we engineered mutant mouse lines PkmM1/M1 or PkmM2/M2, each expressing only PKM1 or PKM2, by knocking in (KI) the Pkm locus using cassettes consisting of the 30 half of human PKM1 or PKM2 cDNA with a poly(A) signal at exon 8 (Figure 1A). Mutant mice appropriately expressed only PKM1 or PKM2 protein and retained tissue/cell-type-specific expression (Figures S1A–S1G). We compared the PKM mRNA levels in PkmM1/M1 or PkmM2/M2 mouse tissues using qRT-PCR (Figure 1B). To do so, we amplified PKM cDNA in a region upstream of the knockin point and common to PkmM1 or PkmM2 alleles. Overall, PKM mRNA levels 356 Cancer Cell 33, 355–367, March 12, 2018

in PkmM1/M1 or PkmM2/M2 mice were comparable in lung, liver, spleen, and small intestine. In brain, however, levels of PKM mRNA in PkmM1/M1were slightly higher than PkmM2/M2, while in muscle they were lower, possibly due to tissue-specific and post-transcriptional regulation of PKM mRNA. We also quantified PKM1 and PKM2 protein levels in brain, small intestine, and muscle of KI mice using mass spectrometry with peptide standards (Matsumoto et al., 2017) (Figure 1C). These analyses revealed that PKM1 and PKM2 protein levels in respective homozygous KI mice closely reflect their mRNA levels, and confirmed that expression of PKM isoforms in KI mice was mutually exclusive. We conclude that PKM1 and PKM2-KI mice express comparable levels of PKM1 and PKM2, respectively, in most tissues. PKM1 Promotes Tumor Growth Cell Autonomously We next used these animals to establish mice with either PKM1 or PKM2 knockin plus Cre-inducible KRASG12D knockin alleles (KrasLSL-G12D) in order to test the effect of PKM isoforms on tumorigenesis. To activate KRASG12D expression we then administered adenovirus-expressing Cre recombinase into tracheas of double KI mice (Figure 1D). Four months later, PkmM1/M1 mice showed enhanced tumor burden relative to wild-type (WT) or PkmM2/M2 mice (Figures 1E and 1F). To assess the effect of PKM isoforms more globally, we next challenged newborn PKM-KI or WT mice with the chemical carcinogen DMBA and evaluated tumor development 8 months later (Figure 1G). PkmM1/M1 mice exhibited a higher incidence of various tumor types than did WT or PkmM2/M2 mice (Figures 1H and 1I). Immunostaining showed that tumors in PkmM1/M1 mice were generally PKM1-positive, except for hepatocellular carcinoma (HCC) in liver and some lung adenocarcinoma cells (Figures 1J and S1H). Liver tumors of PkmM1/M1 mice showed heterogeneous PKM1 expression: some nodules expressed PKM1 and others did not (Figures 1J and S1I). Interestingly, enhanced HCC formation have been reported also in PKM2KO mice, and was explained by a non-cell-autonomous mechanism because those tumor cells express neither PKM1 nor PKM2 (Dayton et al., 2016a). In contrast, we observed PKM1-positive HCC nodules and several other types of tumor-expressing PKM1, implying a possible tumor cell-intrinsic action of PKM1 in PkmM1/M1 mice. To assess a potential cell-autonomous function of PKM1 in tumorigenesis, we established mouse embryonic fibroblasts (MEFs) from WT, PkmM1/M1, or PkmM2/M2 mice, immortalized each with SV40 large-T, and then transformed them with KRASG12V or EGFRex19del (MEF-KRASG12V and -EGFRex19del cells, Figure 2A). The KRASG12V used here differs from the G12D mutant used in animal experiments but retains comparable oncogenic function (Stolze et al., 2015; Hammond et al., 2015). Expression of PKM mRNAs in MEFs and MEF-KRASG12V cells was similar between PkmM1/M1 and PkmM2/M2 groups, although PKM mRNA and protein levels in MEF-EGFRex19del and MEF cells differed somewhat (Figures 2B and 2C). Also, expression levels of respective transduced oncogenes in MEF-KRASG12V and -EGFRex19del cells were indistinguishable between genotypes (Figures S2A–S2C). Proliferation of all lines in vitro was similar among genotypes (Figure 2D). However, when we transplanted cells into nude mice, PkmM1/M1 tumors

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Figure 1. PKM1 Promotes Tumor Growth Cell Autonomously (A) Diagram of Pkm-knockin mice. (B and C) Relative expression of PKM mRNA (sum of PKM1 and PKM2 mRNA) (B) and absolute protein levels of PKM1 or PKM2 (C) in indicated tissues from homozygous PKM-knockin mice. SI, small intestine; GM, gastrocnemius muscle. n = 3 or 5 mice. **p < 0.01, ***p < 0.001; Student’s t test. (D) Experimental design showing KrasG12D-induced lung tumorigenesis. (E) H&E staining of mouse lung 4 months after Ade-Cre administration. Shown are representative lungs (five lobes from same mice) of each genotype. Scale bars, 1 mm. (F) Quantification of tumor area of lesions in mouse lung. n = 9 wild-type (WT), 9 PkmM1/M1 and 6 PkmM2/M2 mice. *p < 0.05; one-way ANOVA with Tukey’s post-hoc test. (G) Timeline of DMBA-primed tumorigenic model. (H) Tumor incidence in WT, PkmM1/M1 or PkmM2/M2 mice challenged with DMBA at post-natal days 3–4. Results are from male mice only. Each row represents one mouse, and filled boxes indicate tumor incidence, with numbers of tumor/nodule(s) in respective organs. HCC, hepatocellular carcinoma; LUAD, lung adenocarcinoma; SCC, squamous cell carcinoma. (I) The number of tumors seen per animal. **p < 0.01; one-way ANOVA with Tukey’s post-hoc test. (J) Immunostaining using anti-PKM1 or anti-PKM2 antibodies of DMBA-induced HCC and SCC tumors in PkmM1/M1 mice. Scale bars, 0.1 mm. All data are mean ± SEM. See also Figure S1.

grew more rapidly than did PkmM2/M2 or WT tumors in both KRASG12Vor EGFRex19del models (Figures 2E–2G). Similar results were seen in experiments with lung epithelial (LE) cells established from PkmM1/M1 and PkmM2/M2 mice and then transformed either with KRASG12V or EGFRex19del (Figures 2A–2C, 2H–2J, S2D, and S2E). Together with autochthonous models, these results indicate that PKM1, rather than PKM2, promotes tumor growth in cell-autonomous manner.

PKM1 Activates Central Carbon Metabolism PKM1 constitutively forms an active tetramer, while PKM2 is activated by allosteric factors such as fructose 1,6-bisphosphate (FBP), a glycolytic intermediate (Figure 3A). PK activity in PkmM1/M1 cells was relatively high, even before glucose stimulation (Figure 3B). By contrast, glucose strongly stimulated PK activity in PkmM2/M2 cells, as expected, although activity remained lower than that seen in PkmM1/M1 cells. Cancer Cell 33, 355–367, March 12, 2018 357

Figure 2. PKM1 Promotes Tumor Growth in Transplantation Models (A) Immunoblots of MEF and LE lysates derived from cells of PkmM1/M1, PkmM2/M2, or WT mice, then transformed with KRASG12V or LE-EGFRex19del. (B and C) Relative expression of PKM mRNA (sum of PKM1 and PKM2 mRNA) (B) and protein levels of PKM1 or PKM2 relative to GAPDH (C) in PkmM1/M1, PkmM2/M2 MEFs, or LE cells. n = 3 or 4 independent lines. *p < 0.05; Student’s t test. (D) Proliferation of PkmM1/M1 or PkmM2/M2 MEF-KRASG12V cells in vitro. Data represent the mean of three independent lines per indicated genotype. (E) MEF-KRASG12V cells were transplanted subcutaneously (s.c.) into nude mice and then tumor volume was measured for 25 days. n = 16 tumors from 4 independent PkmM1/M1 or WT MEFs; n = 12 tumors derived from 3 independent PkmM2/M2 MEFs. **p < 0.01 relative to PkmM2/M2, ###p < 0.001 relative to WT. (F) Volume of individual tumors formed by MEF-KRASG12V cells 18 days after s.c. inoculation into nude mice. Four independent lines of PkmM1/M1 and WT MEFs and three independent lines of PkmM2/M2 MEFs were analyzed in quadruplicate. **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s post-hoc test. (G) Volume of individual tumors formed by MEF-EGFRex19del cells 17 days after s.c. inoculation into nude mice. Tumor volume was measured as in (F). Shown are the size of 32 tumors of 4 independent PkmM1/M1 MEFs and 24 tumors of three independent PkmM2/M2 MEFs. ***p < 0.001; Student’s t test. (H) Proliferation of PkmM1/M1 or PkmM2/M2 LE-KRASG12V cells in vitro. Data represent the mean of three independent lines per indicated genotype. (I) LE-KRASG12V cells were transplanted s.c. into nude mice, and tumor volume was measured. n = 12 tumors from 3 independent PkmM1/M1 or PkmM2/M2 LEs. **p < 0.01, ***p < 0.001; Student’s t test. (J) LE-EGFRex19del cells were transplanted s.c. into nude mice, and tumor volume was measured. n = 12 tumors from 3 independent PkmM1/M1 or PkmM2/M2 LEs. ****p < 0.0001; Student’s t test. All data are mean ± SEM. See also Figure S2.

WT cells showed activity intermediate between PkmM1/M1 and PkmM2/M2. 13 C-Labeled glucose tracer experiments (Figure 3C) indicated that glucose was converted to lactate more rapidly and robustly in PkmM1/M1 than in WT or PkmM2/M2 cells (Figures 3D, 3E, and S3A). Also, in these experiments, levels of 13C-labeled tricarboxylic acid (TCA) intermediates tended to be, or were, significantly increased in PkmM1/M1 compared with WT or PkmM2/M2 cells (Figures 3D and S3A). Consistent with increased flux of glucose to 358 Cancer Cell 33, 355–367, March 12, 2018

lactate, levels of total lactate in PkmM1/M1 cells tended to be higher than those in PkmM2/M2 cells after a change in the medium (0.5 hr in Figure 3F), although that difference was not statistically significant, and changes in lactate levels were transient. It is important to note, however, that intracellular lactate levels at later time points (24 hr in Figure 3F) and net lactate production by PkmM1/M1 cells during this period (Figure 3G) were lower than those of PkmM2/M2 cells, in accordance with a previous report (Christofk et al., 2008). Levels of glycolytic

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intermediates transiently increased immediately after 13C-labeled glucose loading and were comparable between PkmM1/M1 and PkmM2/M2 cells, while G6P levels in PkmM1/M cells tended to be higher than those of PkmM2/M2 cells (Figure S3B). In contrast to previous reports (Lunt et al., 2015), incorporation of 13C from glucose into purine nucleotides, serine and glycine did not decrease in PkmM1/M1 cells. The discrepancy between the flux of glucose to lactate and the total lactate levels in PkmM1/M1 and PkmM2/M2 cells implies that other carbon source(s) contribute to lactate production in tumor cells expressing PKM2, particularly at later phases.

Figure 3. PKM1 Activates Central Carbon Metabolism (A) Catalytic regulation of PKM isoforms. PKM1 forms an active tetramer constitutively, while PKM2 is activated by allosteric factors. (B) PK activities in WT, PkmM1/M1, or PkmM2/M2 MEF-KRASG12V cells starved for glucose and then refed. Shown are mean with ranges of two independent MEF lines per genotype. (C) Illustration of fates of uniformly labeled glucose ([U-13C]glucose)-derived carbons in glycolysis and the TCA cycle. PKM catalyzes the PEP-topyruvate reaction. (D) Levels of [U-13C]glucose-derived isotopomers of lactate (left) and succinate (right) at the times indicated. n = 4 or 6 biological replicates from 2 or 3 independent MEF lines for each genotype. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s post-hoc test. (E) Enrichment of [13C]glucose-derived lactate (calculated as a percentage of total lactate) in MEF-KRASG12V. n = 4 or 6 biological replicates from two or three independent MEF lines for each genotype. **p < 0.01, ***p < 0.001 relative to PkmM2/M2. ###p < 0.001 relative to WT; one-way ANOVA with Tukey’s post-hoc test. (F) Levels of intracellular lactate in MEF-KRASG12V cells at the times indicated after medium change. n = 6, 8, or 9 biological replicates from 3 or 4 independent MEFs for each genotype. *p < 0.05; Student’s t test. (G) Lactate release of MEF-KRASG12V cells at 24 hr after medium change. n = 6 biological replicates from 3 independent MEFs for each genotype. *p < 0.05; Student’s t test. (H) Glutamine (left) and glucose (right) consumed up to the times indicated after medium change. Data were normalized for cellular protein amounts. n = 9 biological replicates from 3 independent MEFs for each genotype. *p < 0.05; Student’s t test. (I) Illustration of fates of [U-13C]glutamine-derived carbons after entry into the TCA cycle. (J) Enrichment of [13C]glutamine-derived lactate, alanine and aspartic acid (calculated as a percentage of total amounts of each metabolite) at 6 hr after tracer-loading. **p < 0.01; Student’s t test (F–H and J). All data are mean ± range (B) or SEM (D–H and J). See also Figure S3.

Accordingly, PkmM2/M2 cells consumed more glutamine than did PkmM1/M1 cells, while glucose consumption by PkmM1/M1 and PkmM2/M2 cells was indistinguishable (Figure 3H). Moreover, tracer experiments using 13C-labeled glutamine (Figure 3I) showed that PkmM2/M2 cells exhibited higher enrichments of 13C-labeled lactate, alanine and aspartic acid than PkmM1/M1, suggesting that PkmM2/M2 cells metabolized glutamine to them more robustly than PkmM1/M1 did (Figures 3J and S3C). Moreover, whole metabolome (Table S1) and pathway analyses showed little impairment of any biosynthetic pathways in Cancer Cell 33, 355–367, March 12, 2018 359

PkmM1/M1 cells at steady state, even after phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) blockade by BEZ235 inducing metabolic stress (Liu et al., 2009) (Figures S3D and S3E). Overall, we conclude that, relative to PKM2, PKM1 more robustly activates glucose catabolism without interfering with other biosynthetic pathways, whereas PKM1 attenuates glutamine metabolism (Figure S3F). To determine whether the in vitro flux analysis described above reflects metabolism occurring in PkmM1/M1 and PkmM2/M2 tumors in vivo, we injected mice bearing tumors derived from MEF-KRASG12V cells with 13C-labeled glucose when tumors were 0.5–1.0 cm in diameter (Figure S3G). As PkmM2/M2 tumors grow more slowly than PkmM1/M1 tumors, more time was required for PkmM2/M2 tumors to reach that size (Figure S3H). One hour after injection, both genotypes showed comparable 13C labeling of lactate, citrate, and malate in tumors (Figure S3I). However, the fumarate/succinate ratio in PkmM1/M1 tumors was significantly reduced compared with that in PkmM2/M2 tumors (Figure S3J), suggesting that PkmM1/M1 tumors are less perfused, as conversion of succinate to fumarate in the TCA cycle requires oxygen (Figure S3K). Given these apparently different perfusion states, we could not further evaluate metabolic fluxes in KI tumors. Active Autophagy Confers Metabolic Advantages To PkmM1/M1 Tumor Cells Next we examined oxygen consumption rates (OCRs) of WT, PkmM1/M1, and PkmM2/M2 cells. Despite a 2- to 3-fold increase in entry of glucose-derived carbon into the TCA cycle (Figures 3D and S3A), increases in OCR in PkmM1/M1 cells (versus WT or PkmM2/M2) were modest in MEF-KRASG12V and not seen in LE-KRASG12V cells (Figures S4A and S4B). These discrepancies may be explained in part by decreased glutamine consumption by PkmM1/M1 cells, as described above. We also hypothesized that mitochondrial integrity might differ between genotypes, given that O2 consumption can occur in dysfunctional mitochondria due to proton leakage (Brand and Nicholls, 2011; Divakaruni and Brand, 2011). Thus, we evaluated mitochondrial phenotypes in PkmM1/M1 and PkmM2/M2 LE-KRASG12V cells. Surprisingly, PkmM2/M2 cells exhibited greater amounts of mitochondria than did PkmM1/M1 cells (Figure 4A), although the mitochondrial membrane potential of PkmM2/M2 cells was significantly lower than that observed in PkmM1/M1 cells, suggestive of mitochondrial dysfunction (Figure 4B). Consistently, mitochondrial and cellular levels of ROS in PkmM2/M2 cells were significantly higher than in PkmM1/M1 cells (Figures 4C and 4D), suggesting that PkmM2/M2 cells accumulate dysfunctional mitochondria. Given that autophagy maintains mitochondrial quality by eliminating damaged mitochondria (i.e., mitophagy [Mizushima and Komatsu, 2011]), we asked whether PkmM1/M1 cells exhibit higher autophagic activity relative to PkmM2/M2 cells. Consistently, levels of free amino acids increased in PkmM1/M1 LE-KRASG12V cells relative to PkmM2/M2 LE-KRASG12V cells (Sou et al., 2008) (Figure 4E). Similar results were obtained in MEF-KRASG12V cells treated with BEZ235 to induce autophagy and in MEF-KRASG12V cell-derived tumors (Liu et al., 2009; Viale et al., 2014) (Figure S4C). PkmM1/M1 cells showed increased conversion of LC3-I to LC3-II following bafilomycin treatment compared with PkmM2/M2 cells (Figure 4F). Assays using a recently developed 360 Cancer Cell 33, 355–367, March 12, 2018

fluorescent autophagic-probe (Kaizuka et al., 2016) also indicated enhanced autophagic flux in PkmM1/M1 relative to PkmM2/M2 cells (Figure S4D). Collectively, these results indicate autophagy is more active in PKM1-expressing cells than in PKM2-expressing cells. To assess the role of autophagic activity in PKM knockin settings, we deleted Atg7, which encodes a factor essential for autophagic activity, in PkmM1/M1 and PkmM2/M2 MEF-KRASG12V cells (Figure S4E). Atg7-KO cells showed virtual loss of ATG5ATG12 conjugate and markedly reduced levels of LC3-II, both of which indicate defective autophagy in these cells. Atg7-KO cells also showed increased mitochondrial mass, lower mitochondrial membrane potential, and higher levels of mitochondrial ROS (Figures S4F–S4H). Furthermore, PkmM1/M1;Atg7-KO MEF-KRASG12V cells showed decreased tumor growth relative to corresponding Atg7 WT cells, while comparable PkmM2/M2 cells showed similar phenotypes (Figure 4G). To determine how PKM1 activity enhances autophagy, we investigated phosphorylation levels of mTOR- and AMP-activated protein kinase (AMPK) substrates, energy charge, AMP levels, and adenosine and adenine secretion, and none of these activities were correlated with active autophagy in PkmM1/M1 cells (Figures S4I–S4L). Overall, these results strongly suggest that PKM1 promotes tumor growth in part through autophagy/mitophagy activated by a mechanism other than mTOR suppression or AMPK activation (Figure 4H). Human Lung Neuroendocrine Tumors Express PKM1 Most tumor cells and their cells/tissues of origin express PKM2 (Bluemlein et al., 2011; Dayton et al., 2016a). Given that the cell of origin likely influences pathway preferences in tumor cells (Mayers et al., 2016; Yuneva et al., 2012), we asked whether tumors arising from PKM1-positive cells expressed PKM1. To do so, we evaluated pulmonary neuroendocrine (NE) tumors (NETs). Pulmonary NETs include a spectrum of tumors from the lowgrade carcinoid to high-grade large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung cancer (SCLC). Importantly, NETs reportedly originate from NE cells, which differentiate from pulmonary epithelial cells in the bronchus (Asselin-Labat and Filby, 2012; Morimoto et al., 2012; Noguchi et al., 2015). Histological analyses of normal mouse lung revealed significant co-localization of CGRP (a pulmonary NE cell marker) with PKM1-positive aggregates in the bronchial epithelium (Figures 5A and S5A). Other epithelial cells were PKM1-negative/ PKM2-positive. When we compared PKM1 and PKM2 mRNA expression in clinical lung NET samples relative to samples of other lung cancers (Figure 5B), NET specimens showed markedly higher PKM1 mRNA levels and a higher PKM1/PKM2 ratio than did non-NET tumors (Figures 5C and S5B). This pattern was also seen in vitro in cultures of NET-derived cell lines (all of the SCLC type) and in xenograft tumors obtained by transplantation of cell lines into immunocompromised mice (Figures 5D, 5E, S5C, and S5D). Analysis using a pan-cancer cell line panel confirmed that the SCLC PKM1/PKM2 mRNA ratio was higher when compared with various other cancers (Figure S5E). The SCLC cell lines expressed substantial amount of PKM1 protein, while it varied in cell lines examined (Figures 5F and S5F). We next quantified PKM1 and PKM2 protein levels in five SCLC and two non-NET lung cancer cell lines using mass

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Figure 4. Active Autophagy Confers Metabolic Advantages to PkmM1/M1 Tumor Cells (A) Mitochondrial mass of LE-KRASG12V cells. n = 18 biological replicates from 3 independent LE lines per indicated genotype. (B) Mitochondrial membrane potential of LE-KRASG12V cells was estimated by assessing the ratio of MitoTracker Deep Red to MitoTracker Green. Results are shown as averages of three independent LE lines per indicated genotype. (C and D) Mitochondrial (C) and cellular (D) ROS levels in LE-KRASG12V cells. n = 18 biological replicates from three independent LE lines per indicated genotype. (E) Free L-amino acid levels in LE-KRASG12V cells. Results are shown as averages of three independent LE lines per indicated genotype. (F) Anti-LC3 immunoblot showing PkmM1/M1 or PkmM2/M2 MEF-KRASG12V cells treated with bafilomycin-A1 for 6 hr or left untreated. Shown are representative analyses of two independent MEF lines per genotype. (G) Parental and Atg7-deficient PkmM1/M1 and PkmM2/M2 MEF-KRASG12V cells were transplanted s.c. into nude mice, and tumor volume was measured. Five clones were analyzed for PkmM1/M1 and PkmM2/M2 cells. n = 6 and 4 tumors from parental lines and Atg7-KO clones, respectively. (H) Model of how PKM1-dependent autophagy promotes tumor growth. All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Student’s t test. See also Figure S4.

spectrometry with peptide standards (Matsumoto et al., 2017) (Figure S5G). The absolute ratio of PKM1 versus total PKM was 16%–38% in SCLCs and <2% in non-NETs (Figure 5G), although PKM1 was not the major isoform even in SCLCs. However, we also found that, when overexpressed, PKM1 and PKM2 can form a complex in intact cells (Figures S6A and S6B). Interestingly, co-expression of PKM1 increased the tetramer/monomer ratio of PKM2 dose dependently (Figure S6C). Induction of tetrameric forms of PKM containing PKM2 was also seen when

PKM1 expression increased due to either heterozygous PKM1 knockin in embryonic stem cells (Figure S6D) or by knockdown in HeLa cells of splicing factors (PTBP1, hnRNPA1, and hnRNPA2) that negatively regulate PKM1 pre-mRNA splicing (Figures S6E and S6F) (David et al., 2010). Furthermore, addition of purified PKM1 to purified PKM2 in vitro reduced PKM2 monomer levels and increased levels of PKM2 tetramers, as did addition of FBP, an allosteric activator (Figures 6A and S6G). Importantly, in the absence of FBP, combined activities Cancer Cell 33, 355–367, March 12, 2018 361

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Figure 5. Human Lung NETs Express PKM1 (A) Immunohistochemical staining of normal bronchus of adult WT mice. Shown are two sets of serial sections. Arrows indicate respective PKM1high, PKM2low, or CGRP+ aggregates within the respiratory epithelium. Scale bars, 25 mm. (B) Relative expression of NET markers in a lung cancer cohort were examined by qRT-PCR and are shown by a color scale. Each line represents one patient. Ad, adenocarcinoma; Sq, squamous carcinoma; Ad/Sq, adeno-squamous carcinoma; LCNEC, large-cell neuroendocrine carcinoma; NEC, neuroendocrine carcinoma; SCLC, small-cell lung cancer; TC, typical carcinoid; and WDNEC, well-differentiated neuroendocrine carcinoma. (C) PKM1/PKM2 mRNA ratios in NET (n = 17) compared with ratios seen in other types of lung cancer (n = 35). (D) Unsupervised clustering of human lung cancer cell lines based on mRNA expression. Distinct tumor types can be differentiated by NET marker transcripts. Expression of mRNA encoding PKM isoforms in cell lines is shown at the bottom. (E) PKM1/PKM2 mRNA ratios in SCLC (n = 13) compared with ratios seen in non-NET cell lines (n = 4). (F) Immunoblots of indicated SCLC and non-NET lines. (G) Absolute ratio of PKM1 to total PKM proteins in lung cancer cell lines, as determined by mass spectrometry. Data in (C and E) are represented as mean ± SEM. *p < 0.05, **p < 0.01; Student’s t test. See also Figure S5.

of PKM1/PKM2 varied exponentially, rather than linearly, relative to the amount of PKM1 (Figure S6H). Km and Vmax values of a 1:1 PKM1/PKM2 mixture were similar to those of PKM1 or PKM2 in the presence of FBP (Figures S6I–S6K). These results strongly 362 Cancer Cell 33, 355–367, March 12, 2018

suggest that the presence of PKM1 promotes inclusion of PKM2 in an active tetramer, even in the absence of an allosteric factor (Figure S6L; see Discussion for details). Importantly, when cells were cultured in low glucose, PKM2 seen in SCLC cells

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Figure 6. PKM1 Activates PKM2 and Is Required for SCLC Cell Proliferation (A) Complex formation of purified PKM1 and PKM2 in vitro, as analyzed by blue native (BN) PAGE followed by CBB staining. FBP, fructose 1,6-bisphosphate. (B) Tetramer formation of PKM1 and PKM2 in lung cancer cell lines cultured in low glucose (1 mM) medium for 1 day, as analyzed by BN-PAGE. Anti-GAPDH (forming homotetramer [Jenkins and Tanner, 2006]) blot served as loading controls. Asterisk indicates residual signal of anti-PKM1 blot. (C) PK activities of the lysates in (B) were measured with or without FBP (left). The activity ratio of each cell line (without FBP) relative to FBP-revealed activity (defined as ‘‘potential activity’’) was calculated and compared between SCLC and non-NET cell lines (right). Data are represented as mean ± SEM. ***p < 0.001; Student’s t test. (D) Schematic showing functional analysis of PKM1 or PKM2 in SCLC. (E) Immunoblots of 87-5 cells expressing exogenous Flag-tagged PKM1 or PKM2 before and after infection with virus expressing small hairpin RNA (shRNA) targeting endogenous PKM. (F) 87-5 and Lu139 cells engineered to express mPKM1 or mPKM2 were infected with lentivirus expressing shRNA targeting endogenous PKM. Viable cells were counted and passaged every 3 to 4 days. See also Figure S6.

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resided primarily in an active tetramer, which was not the case when we used non-NET cells (Figures 6B and 6C). To determine the biological relevance of PKM1 expression in SCLC, we transduced the SCLC line 87-5 with exogenous mouse PKM1 or PKM2 and knocked down endogenous human PKM (of either isoform) with small hairpin RNA. Cells expressing mouse PKM1 and PKM2 are designated 87-5-mPKM1 and 87-5mPKM2 cells, respectively (Figures 6D and 6E). 87-5-mPKM2 cells showed markedly reduced proliferation compared with 87-5-mPKM1 cells (Figure 6F). Similar results were obtained in the SCLC line Lu139 expressing exogenous mouse PKM1 or PKM2, then endogenous PKM was knocked down (Figure 6F). These results indicate that PKM2 cannot substitute for PKM1 in supporting SCLC proliferation. DISCUSSION Here we show that expression of PKM1, rather than PKM2, activates glucose metabolism and promotes tumor growth cell autonomously in a KrasLSL-G12D mouse model and in KRASG12V MEFs. We propose that activation of autophagy/mitophagy confers a metabolic advantage mediated by PKM1, favoring malignancy. We also show that human pulmonary NETs express PKM1 at significant levels, and that this expression is required for SCLC proliferation. Our results, however, do not exclude the possibility that PKM2 expression may confer some advantage(s) to tumor cells in a non-cell-autonomous manner or in other cell types. Nevertheless, our observations provide an alternate mechanism for accelerated tumorigenesis reported in PKM2-deficient mice, a phenotype that has been explained thus far by non-cell-autonomous mechanisms thorough dysregulation of systemic glucose homeostasis (Dayton et al., 2016a). It is now recognized that tumor cells or tissues of origin influence the molecular characteristics of a tumor (Mayers et al., 2016; Yuneva et al., 2012). Selection of a particular PKM isoform in tumors supports this concept. We and others have shown that cells/tissues of origin of most tumor types (among them, epithelial cells for carcinomas, or neural stem or progenitor cells and astrocytes for glioma) express PKM2 prior to neoplastic transformation, regardless of proliferation status (Bluemlein et al., 2011; Dayton et al., 2016a; Tech et al., 2017). By contrast, pulmonary NETs and their cells of origin (NE cells in the bronchus) both exhibit relatively high PKM1 expression, supporting the hypothesis that PKM isoform selection in tumors mirrors that seen in the tissue of origin. Thus, it is possible that expression of PKM2 (and not of PKM1) in normal cells may have an anti-cancer effect, as genetic replacement of PKM2 with PKM1 in some mouse models enhances tumor development, as shown here and reported previously (Dayton et al., 2016a; Israelsen et al., 2013; Tech et al., 2017). Exogenous PKM1 expression has, however, been reported to suppress tumor-forming activity of mouse mammary tumor cells in a transplantation model (Israelsen et al., 2013). Although Cre activation alone mediates DNA damage and represses growth of primary MEFs (Loonstra et al., 2001), Cremediated PKM1 expression from the mutant Pkm allele (Pkm2flox) reportedly slowed proliferation of MEFs to a greater extent than did Cre activation (Lunt et al., 2015). These findings suggest that limiting PKM activity confers growth advantage(s) to proliferating cells. However, analysis of our knockin model 364 Cancer Cell 33, 355–367, March 12, 2018

indicates that PKM1, which is a constitutively active isoform, is compatible with cell proliferation. Considered together with the observation that mice lacking both PKM1 and PKM2 die in early embryogenesis (R. Tanaka et al., unpublished data), we propose that higher PKM activity overall promotes, rather than inhibits, cell proliferation cell autonomously, although this outcome may vary in some cell lines or under specific conditions. Lung NETs, including LCNEC, NEC, and SCLC, account for 15%–20% of human lung cancers, and patient prognosis in these cases is poorer than that of patients with non-NET lung cancers. Although recent genomic studies show simultaneous biallelic inactivation of TP53 and RB1 in human SCLC, these studies reveal few driver mutations in SCLC that could be therapeutically targeted (George et al., 2015). Here we provide a rationale for targeting PKM1, possibly by small molecules to inhibit its activity, tetramer formation, or PKM1-PKM2 splicing, in this context. While the proportion of PKM1 in total PKM was 16%–38% in SCLCs examined, it is noteworthy that PKM1 promotes incorporation of PKM2 into an active tetrameric form, suggesting that an increase in the cellular PKM1/PKM2 ratio might alter cellular PK activity in a non-linear manner, as we observed in vitro. Based on observed exponential changes in activity in vitro following addition of PKM1 to PKM2, we propose that PKM1 stimulates PKM2 by promoting formation of a hetero-complex. Accordingly, expression of PKM1 in SCLC cells may boost PKM2 activity in a factor-independent manner via direct interaction. Here, we show that PKM1 activates glucose catabolism. Importantly, our tracer experiments demonstrate that PKM1, relative to PKM2, enhances not only entry of glucose-derived carbons into the TCA cycle but also glucose conversion to lactate. These results challenge the model that PKM2 limits glucose oxidation but enhances its fermentation (Christofk et al., 2008). Instead, our results strongly argue that PKM2 limits both due to inefficient conversion of PEP to pyruvate, a step that constitutes a hub for the TCA cycle, and is necessary for lactate production. Nevertheless, net lactate production is downregulated in a PKM1-knockin (PKM1-KI) setting, suggesting that other carbon source(s) contribute to lactate production, particularly in tumor cells expressing PKM2, although the extent may vary according to cell context. In this regard, glutamine is a strong candidate, since it is reported that 60% of glutamine consumption can be accounted for by its conversion to alanine and lactate in a glioblastoma cell line (DeBerardinis et al., 2007). Strikingly, our tracer experiments showed that PkmM1/M1 cells contain less glutaminederived lactate, alanine, and aspartic acid than do PkmM2/M2 cells. Given decreased glutamine consumption by PkmM1/M1 cells, glutaminolysis may be impaired under the active flux of glucose-derived carbons into the TCA cycle dependent on PKM1. Metabolic re-wiring by PKM1 may eventually decrease net lactate production, although detailed mechanisms underlying this activity remain unknown. We performed tracer experiments in mice bearing allograft tumors, but could not fully evaluate metabolic flux in tumors apparently due differing vascular perfusion. The lower fumarate/succinate ratio seen in PkmM1/M1 tumors suggests that those tumors are more hypoxic than PkmM2/M2 tumors. It is possible that the vasculature cannot keep up with the rapid growth of PkmM1/M1 tumor cells. Due to these complications, it is currently difficult to assess potential metabolic difference(s) between PKM1- and PKM2-expressing

tumor cells based on these tracer experiments. Thus, we do not rule out the possibility that metabolic fluxes governed by PKM1 and PKM2 may differ in vitro and in vivo. Future studies should clarify whether metabolic phenotypes of PkmM1/M1 and PkmM2/M2 cells observed in vitro reflect those in vivo. We observed that PKM1-expressing cells exhibit more active autophagy than PKM2-expressing cells, suggesting that PKM1 drives active glucose metabolism and autophagy in tumor cells. Generally, autophagic activity is inversely correlated with the availability of nutrients, particularly amino acids, or a cellular high energy state (Mizushima and Komatsu, 2011). Neither ATP levels nor amino acid levels were downregulated in PKM1-KI cells, but were rather upregulated. However, it is also known that autophagy is a highly ATP-dependent process: for example, formation of the ATG5-ATG12 conjugate and LC3-II, both catalyzed by ATG7, requires ATP hydrolysis (Ichimura et al., 2004; Mizushima et al., 1998). Given that ATP is required for autophagy, unusually high ATP levels in a PKM1-KI context may boost basal autophagy by an as yet unidentified mechanism, although lower energy status is also reportedly linked to autophagy via the AMPK-Ulk1 axis (Mizushima and Komatsu, 2011). Future studies should address mechanisms by which PKM1 activates autophagy. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCES SHARING EXPERIMENTAL MODELS AND SUBJECT DETAILS B Mice B MEFs and LE Cells B Human Lung Cancer Samples B Human Cell Lines METHOD DETAILS B Generation of Isoform-Specific PKM Antibodies B qRT-PCR Analyses B Absolute Quantification of PKM Proteins B KRAS-Driven Lung Tumorigenesis in Mice B Carcinogen Tests in Mice B IF and IHC Analyses B Isolation, Expansion and Transformation of LE Cells B Proliferation Assay In Vitro B Transplantation Experiments B PK Activity Assay B Enzymatic Quantification of Metabolites B Metabolite Measurement by Mass Spectrometry B Tracer Labeling Experiments In Vitro B Tracer Labeling Experiments In Vivo B Measurement of Oxygen Consumption Rates B Mitochondria and ROS Assays B Generation of Atg7-Knockout Cells B Autophagic Probe Assay B Transfection Experiments B Blue-Native PAGE Analysis B SCLC Cells Expressing Exogenous PKM1 or PKM2 QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and two tables and can be found with this article online at https://doi.org/10.1016/j.ccell.2018.02.004. ACKNOWLEDGMENTS We acknowledge Dr L. Cantley, Dr M.G. Vander Heiden, Dr D. Anastasiou, Dr W. Haln, Dr D. Trono, Dr S. Yamanaka, and Dr N. Mizushima for providing plasmid constructs, and Dr J. Takeda for providing KY1.1 embryonic stem cells. Thanks are also due to members of the Pathology facility of Miyagi Cancer Center Hospital and the Tissue bank Center for technical help in tissue analyses, and Ms Y. Chiba, Ms M. Oh-uchi for secretarial assistance, and Dr E. Lamar for English editing. This work was supported by JSPS KAKENHI grants (24501326, 16K14621 and 17K19620) to N.T., 26430130 to H.S., 17K07231 and 16K07187 to S.I., 15K14387 to I.S., and 16K10486 to K.M., the Takeda Foundation to N.T., the Mochida Memorial Foundation for Medical and Pharmaceutical Research to N.T., the Kato Memorial Bioscience Foundation to N.T., the Uehara Memorial Foundation to N.T., and the Sagawa Foundation for Promotion of Cancer Research to N.T. AUTHOR CONTRIBUTIONS N.T. designed the experiments. M. Morita, T.S., M.N., Y. Sakamoto, Y.I., R.T., K.K., K.Y., Y. Sugiura, H.T., M.K., H.W., G.K., S.M., A.K., M.O., M. Matsumoto, M.K., T.W., T.S., H.S., and N.T. performed the experiments. M. Morita, Y. Sugiura, S.I., Y.Y., R.K., I.S., Y.O., T.F., H.M., M.S., K.I.N., T.S., M. Maemondo, and N.T. analyzed the data. M. Morita and N.T. wrote the paper. DECLARATION OF INTERSTS The authors declare no competing financial interests. Received: September 6, 2017 Revised: December 28, 2017 Accepted: February 6, 2018 Published: March 12, 2018 REFERENCES Anastasiou, D., Poulogiannis, G., Asara, J.M., Boxer, M.B., Jiang, J.K., Shen, M., Bellinger, G., Sasaki, A.T., Locasale, J.W., Auld, D.S., et al. (2011). Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334, 1278–1283. Asselin-Labat, M.L., and Filby, C.E. (2012). Adult lung stem cells and their contribution to lung tumourigenesis. Open Biol. 2, 120094. Bluemlein, K., Gruning, N.M., Feichtinger, R.G., Lehrach, H., Kofler, B., and Ralser, M. (2011). No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2, 393–400. Brand, M.D., and Nicholls, D.G. (2011). Assessing mitochondrial dysfunction in cells. Biochem. J. 435, 297–312. Chaneton, B., and Gottlieb, E. (2012). Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in cancer. Trends Biochem. Sci. 37, 309–316. Chaneton, B., Hillmann, P., Zheng, L., Martin, A.C.L., Maddocks, O.D.K., Chokkathukalam, A., Coyle, J.E., Jankevics, A., Holding, F.P., Vousden, K.H., et al. (2012). Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491, 458–462. Christofk, H.R., Vander Heiden, M.G., Harris, M.H., Ramanathan, A., Gerszten, R.E., Wei, R., Fleming, M.D., Schreiber, S.L., and Cantley, L.C. (2008). The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230–233. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823.

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Viale, A., Pettazzoni, P., Lyssiotis, C.A., Ying, H., Sanchez, N., Marchesini, M., Carugo, A., Green, T., Seth, S., Giuliani, V., et al. (2014). Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function. Nature 514, 628–632. Ward, P.S., and Thompson, C.B. (2012). Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate. Cancer Cell 21, 297–308. Yamamoto, T., Takano, N., Ishiwata, K., Ohmura, M., Nagahata, Y., Matsuura, T., Kamata, A., Sakamoto, K., Nakanishi, T., Kubo, A., et al. (2014). Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway. Nat. Commun. 5, 3480. Yoshimoto, S., Loo, T.M., Atarashi, K., Kanda, H., Sato, S., Oyadomari, S., Iwakura, Y., Oshima, K., Morita, H., Hattori, M., et al. (2013). Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499, 97–101. Yuneva, M.O., Fan, T.W., Allen, T.D., Higashi, R.M., Ferraris, D.V., Tsukamoto, T., Mate´s, J.M., Alonso, F.J., Wang, C., Seo, Y., et al. (2012). The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metab. 15, 157–170.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-LC3

MBL

PM036

Rabbit polyclonal anti-Actin-HRP

MBL

PM053-7

Rabbit polyclonal anti-GAPDH-HRP

MBL

M171-7

Antibodies

Goat polyclonal anti-Pkm

Abcam

Ab6191

Anti-CGRP

Abcam

ab36001

Anti-LC3

CST

12741

Anti-ATG5

CST

12994

Anti-ATG7

CST

8558

Anti-Ki67 mAb

Roche

30-9

Anti-pT389 S6K1

CST

9234

Anti-S6K1

CST

2708

Anti-pT65 4E-BP1

CST

9451

Anti-4E-BP1

CST

9452

Anti-pT757 Ulk1

CST

14202

Anti-pS317 Ulk1

CST

12753

Anti-Ulk1

CST

8054

Anti-pS240/244-S6

CST

9204

Anti-S6

CST

2708

Anti-pT172-Ampka

CST

2535

Anti-Ampka

CST

5832

Anti-Ras

CST

3339

Anti-EGFR

CST

4267

Anti-PTBP1

Abnova

H00005725-M01

Anti-hnRNP a1

Sigma

R9778

Anti-hnRNP a2

Abcam

ab6102

Mouse monoclonal anti-Flag (M2)

Sigma

F3165

Anti-HA

Sigma

11583816001

Anti-Myc tag

MBL

B-AM3185M

Anti-CD45 (mouse)-MicroBeads

Miltenyi

130-052-301

Rabbit polyclonal anti-Pkm1

This study

AB_2721920

Rabbit polyclonal anti-Pkm2

This study

AB_2721921

Rat monoclonal anti-Pkm2

This study

AB_2721922

RIKEN

RDB01748

Human lung cancer tissues

MCC Tissue Bank Ctr

http://www.miyagi-pho.jp/mcc/kenkyu/ tissue-bank.html

Mouse tissues

This study

N/A

[U-13C] glucose

Cambridge Isotope Laboratories

CLM-1396-5

[U-13C] glutamine

Cambridge Isotope Laboratories

CLM-1822-H-0.25

Fugene 6

Promega

E2691

mTRAQ D0

SCIEX

4440015

mTRAQ D4

SCIEX

4427698

Bacterial and Virus Strains Adeno-Cre Biological Samples

Chemicals, Peptides, and Recombinant Proteins

(Continued on next page)

e1 Cancer Cell 33, 355–367.e1–e7, March 12, 2018

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

LipofectAMINE 2000

Thermo Fisher Scientific

11668027

LipofectAMINE RNAi MAX

Thermo Fisher Scientific

13778030

Lamina Propria Dissociation Kit

Miltenyi

130-097-410

Accutase

Merck

SCR005

Mouse EGF

Peprotech

315-09

Matrigel

Corning

#354234

Y27632

Wako

251-00514

MitoTracker Green

Thermo Fisher Scientific

M7514

MitoTracker DeepRed

Thermo Fisher Scientific

M22426

MitoSOX Red

Thermo Fisher Scientific

M36008

CellROX Green

Thermo Fisher Scientific

C10444

FBP

Sigma

47810

BEZ235

Santa Cruz Biotech

sc-364429

Bafilomycin A1

Santa Cruz Biotech

sc-201550A

DMBA

Sigma

D3254

Rotenone

Sigma

R8875

Antimycin A1

Sigma

A8674

Pyruvate Kinase Assay kit

BioVision

K709

HA-tagged Protein PURIFICATION KIT

MBL

3320

Lactate Colorimetric Assay Kit II

BioVision

K627

L-Amino Acid Quantitation Colorimetric/Fluorometric Kit

BioVision

K639

This study

https://doi.org/10.17632/9pvjp45zgn.1

Critical Commercial Assays

Deposited Data CE-MS dataset-Metabolome (43 samples) Experimental Models: Cell Lines Human: PLAT-E cells

Morita et al., 2000

N/A

Human: MS-1 cells

Riken BRC

RCB0725

Human: T3M12 cells

Riken BRC

RCB2281

Human: Lu134A cells

Riken BRC

RCB0466

Human: Lu138 cells

Riken BRC

RCB1785

Human: Lu139 cells

Riken BRC

RCB0469

Human: Lu140 cells

Riken BRC

RCB0470

Human: Lu141 cells

Riken BRC

RCB1772

Human: Lu143 cells

Riken BRC

RCB1773

Human: Lu165 cells

Riken BRC

RCB1184

Human: 87-5 cells

Riken BRC

RCB2092

Human: H209 cells

ATCC

HTB-172

Human: A549 cells

Riken BRC

RCB0098

Human: H1299 cells

ATCC

CRL-5803

Human: HS24 cells

Maemondo et al., 2004

N/A

Human: H1975 cells

ATCC

CRL-5908

Human: MM-8 cells

This study

N/A

Human: 293 cells

Riken BRC

RCB1637

Human: HeLa cells

Riken BRC

RCB0007

Mouse: C57BL/6NJcl

Japan Clea

C57BL/6NJcl

Mouse: Nude: CAnN.Cg-Foxn1nu/CrlCrlj

Charles River Laboratories Japan

BALB/c-nu

Mouse: NOG: NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic

In-Vivo Science

NOD/Shi-scid,IL-2RgKO Jic

Experimental Models: Organisms/Strains

(Continued on next page)

Cancer Cell 33, 355–367.e1–e7, March 12, 2018 e2

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Mouse: KrasLSL-G12D: B6.129S4-Krastm4tyj/J

Jackson Laboratory

008179

Mouse: PkmM1: B6.129-Pkmtm1Ntan(N7)

This study

N/A

Mouse: PkmM2: C57BL/6-Pkmtm3Ntan

This study

N/A

Oligonucleotides Primers for aRT-PCR, see Table S1

This study

N/A

Universal ProbeLibrary Probes, see Table S1

Roche

04684974001

ON-TARGET plus human PTBP1 siRNA

Dharmacon

L-003528-00-0005

ON-TARGET plus human hnRNPa1 siRNA

Dharmacon

L-008221-00-0005

ON-TARGET plus human hnRNPa2 siRNA

Dharmacon

L-011690-01-0005

Addgene #1780

Recombinant DNA pBabe-neo largeT cDNA

Hahn et al., 2002

pBabe-puro Kras-V12

Dr W.C. Hahn Lab

Addgene #9052

pBabe-puro hEGFRex19del

Sato et al., 2017

N/A

pCMV-HA-Pkm1

This study

N/A

pCMV-HA-Pkm2

This study

N/A

pCMV-Myc-Pkm1

This study

N/A

pMRX-IP-GFP-LC3-RFP

Kaizuka et al., 2016

Addgene #84573

pLenti6-UbC-mSlc7a1

Takahashi et al., 2007

Addgene #17224

psPAX2

Dr D. Torono Lab

Addgene #12260

pMD2.G

Dr D. Torono Lab

Addgene #12259

pLHCX-Flag-mPkm1

Christofk et al., 2008

Addgene #44239 Addgene #44240

pLHCX-Flag-mPkm2

Christofk et al., 2008

pLKO-shPkm2_4

Anastasiou et al., 2011

Addgene #42516

pX330-U6-Chimeric_BB-CBh-hSpCas9

Cong et al., 2013

Addgene #42230

pEGFP-C1

Clontech

https://www.clontech.com/

GraphPad prism

GraphPad Software

https://www.graphpad.com/

ImageJ software

Open source

https://imagej.nih.gov/ij/

CRISPR Design tool

Hsu et al., 2013

http://crispr.mit.edu/

Software and Algorithms

CONTACT FOR REAGENT AND RESOURCES SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nobuhiro Tanuma ([email protected]) EXPERIMENTAL MODELS AND SUBJECT DETAILS Mice All animal experiments were performed with approval of the Miyagi Cancer Center Research Institute Animal Care and Use Committee. Unless stated, mouse experiments were done in specific pathogen-free (SPF) facilities. KrasLSL-G12D mice (B6.129S4-Krastm4tyj/J (DuPage et al., 2009)) were obtained from The Jackson Laboratory. PkmM1/+ ES cells (ESCs) were generated by gene-targeting using KY1.1 ESCs (hybrid of B6 and 129 strains). After germline transmission was achieved, heterozygous PkmM1/+ male mice were backcrossed to the C57BL/6NJcl strain for 4 generations using speed-congenic methods. Analysis of an N4 male mouse showed that all 80 short tandem repeat markers were substituted with those of the recipient strain, and thus mice were considered congenic except for sex chromosomes. The N4 male mouse was crossed with C57BL/6NJcl mice to obtain N5 females lacking a Y chromosome. PkmM2/+ ESCs were similarly obtained using RENKA ESCs (BL6 background, Transgenic Co, Kobe, Japan). PkmM2/+ offspring obtained (F0) were crossed with C57BL/6NJcl mice for 3 generations. All experiments using single knock-in mice and cell lines established from those animals were performed using littermates obtained by crossing heterozygous males and females. Phenotypic similarity of Pkm-wild-type mice from the two strains (PkmM1 and PkmM2) was confirmed in every experiment. PkmM1/M1 and PkmM2/M2 mice developed normally and were fertile, and we observed no significant difference between them and WT mice in terms of incidence of spontaneous tumors after one year of observation (data not shown). e3 Cancer Cell 33, 355–367.e1–e7, March 12, 2018

MEFs and LE Cells MEFs obtained from E13.5 embryos were cultured for 2-3 days under hypoxia (3% O2) to prevent possible senescence and then stocked until use. Cell immortalization and transformation were performed at normoxia. Primary MEFs were immortalized for 4 passages (one-tenth dilution per passage) after transfection with SV40 Large-T plasmid using Fugene6 reagent. KRASG12V (a gift of Dr W. Haln via Addgene, # 9052) and EGFRex19del were transduced with retroviral vectors containing Puror as a selection marker. Ecotropic viruses were produced using PLAT-E cells (a gift of Dr T. Kitamura). Primary LE cells were isolated from 8-12 weeks-old female mice and expanded in Matrigel-assisted 3D culture in the presence of EGF (Sato et al., 2017, see ‘‘Method Details’’ section). Transformation of LE cells were performed by simultaneous transduction with SV40 Large-T together with KRASG12V or EGFRex19del, all using retroviral vectors. Human Lung Cancer Samples Human tumor samples (surgical samples collected with informed consent from patients) were obtained from the tumor tissue-bank of Miyagi Cancer Center. The study was reviewed and approved by the Institutional Review Board of Miyagi Cancer Center. Human Cell Lines The human SCLC lines MS-1, T3M12, Lu134A, Lu138, Lu139, Lu140, Lu141, Lu143, Lu165, and 87-5 were provided from Riken Bioresource Center (Tsukuba, Japan). H209 cells were provided by ATCC (Manassas, VA). Human MM8 cells were established in our lab after obtaining informed consent of tissue donors (approved by the review board of Miyagi Cancer Center). All SCLC lines were cultured in RPMI supplemented with 10% FCS. Four NSCLC lines (A549, H1299 and HS24 (Maemondo et al., 2004) or H1975) were provided by ATCC or by JCRB (Ibaraki, Japan). A549 cells were cultured in RPMI supplemented with 10% FCS. H1299, HS24 and H1975 cells were cultured in DMEM supplemented with 10% FCS. All lines were verified as mycoplasma-free using a MycoAlert kit (Lonza, Basel, Switzerland). METHOD DETAILS Generation of Isoform-Specific PKM Antibodies Rabbit polyclonal PKM1 or PKM2 antibodies were generated in our lab against peptides corresponding to amino acid sequences encoded by mutually exclusive exons (LVRASSHSTDLMEAMAMGGV for PKM1, and LRRLAPITSDPTEATAVGAV for PKM2). Similarly, rat monoclonal PKM2 antibody (#4B2) was obtained by immunizing rats with the above PKM2 peptide. One positive hybridoma clone (#4B2) was established and used to obtain conditioned medium followed by antibody purification using ProteinG-Sepharose and a standard procedure. qRT-PCR Analyses RNAs were reverse-transcribed using random primers and Superscript III RTase (Thermo Fisher Scientific, Waltham, MA). qRT-PCR analysis was undertaken using a LightCycler 480, a LightCycler 480 probes master, TaqMan probes from a universal probe library (UPL) (Roche), and primers indicated in Table S2. To analyze PKM1 or PKM2 mRNA levels in human tissues and cell lines, we prepared two sets of primers (Assay Design 1 and 2) and two corresponding TaqMan probes for each PKM mRNA. Unless specified, results obtained using ‘‘Assay Design 1’’ are presented in this paper. Assay Design 2 gave similar results (data not shown). Clustering analyses were performed using GeneSpring software (GE, Fairfield, CT). Absolute Quantification of PKM Proteins Absolute abundance of PKM1 and PKM2 proteins was determined by iMPAQT platform (Matsumoto et al., 2017). In brief, whole tissue/cell digest were labeled with mTRAQD0 (light, (SCIEX, Framingham, MA)), mixed with mTRAQD4 (heavy, (SCIEX))-labeled synthetic peptides, and subjected to multiple reaction monitoring analysis with liquid chromatography coupled triple stage quadrupole mass spectrometer. The targeted peptide sequences used are LFEELVR for PKM1, LAPITSDPTEATAVGAVEASFK for PKM2, and TVDGPSGK for GAPDH, which are selected from iMPAQT-knowledge database (Matsumoto et al., 2017). The absolute abundance of proteins was determined on the basis of the height ratio for light (sample) and heavy (internal standard) peaks in the chromatogram. Experiments using mouse tissues/cells were performed by the service provider Kyusyu Pro Search LLP (Fukuoka, Japan). KRAS-Driven Lung Tumorigenesis in Mice To perform KRAS-driven lung tumorigenesis, PkmM1/+ or PkmM2/+ mice were crossed with KrasLSL-G12D/+ mice. PkmM1/+;KrasLSL-G12D/+ and PkmM2/+;KrasLSL-G12D/+ male and female mice obtained were further crossed to obtain 3-4 PkmM1/M1;KrasLSL-G12D/+, PkmM2/M2;KrasLSL-G12D/+ and Pkm+/+;KrasLSL-G12D/+ male mice. Male mice were then crossed with corresponding PkmM1/M1, PkmM2/M2 and Pkm+/+ females by in vitro fertilization (IVF), and offspring were used for experiments. Adenovirus expressing Cre was administered into the trachea by surgical injection of 1 x 106 pfu/5 ml/mouse in age- (12 weeks after birth) and sex-matched mice.

Cancer Cell 33, 355–367.e1–e7, March 12, 2018 e4

Carcinogen Tests in Mice IVF was performed to obtain mice used for DMBA experiments. Mice at postnatal days 3-4 were painted with 50 ml of 0.5% DMBA in acetone on their dorsal surface (Yoshimoto et al., 2013). IF and IHC Analyses Immuno-histochemical staining was performed using a Ventana instrument and reagents (Roche). Immuno-fluorescence was performed using anti-mouse-IgG-Alexa488, anti-rabbit IgG-Alexa546 and anti-rat IgG-Alexa488 as secondary antibodies, and DAPI. Confocal images were obtained using a Nikon A1 (Nikon, Tokyo, Japan) or Zeiss LSM600 (Carl Zeiss, Jena, Germany) microscope. Upon immuno-staining of tissue sections, antigen-retrieval was not performed except in the case of anti-Ki67 antibody. Isolation, Expansion and Transformation of LE Cells Lungs dissected from mice were washed several times with cold PBS and then treated with tissue-digesting solution (Lamina Propria Dissociation kit, Miltenyi), followed by processing using a ‘‘Gentle MACS’’ cell-isolation system (Miltenyi) according to instructions. After CD45+ cell depletion using anti-CD45-magnetic beads and AutoMACS (Miltenyi), cells were suspended in DMEM with 10% FCS and seeded into 12-well plates that had been pre-loaded with 90 ml polymerized Matrigel. The next day, after removal of medium and floating dead cells, another 90 ml Matrigel was loaded and plates were incubated 40 min at 37 C to sandwich cells within polymerized Matrigel. Wells were then refilled with Advanced DMEM/F12 medium supplemented with 1x penicillin/glutamine/streptomysin, 1x insulin/transferrin/selenium, 10 ng/ml EGF and 5 mM Y27632. Medium was replaced every 3 to 4 days. After 2 to 3 weeks, propagated LE spheres were obtained and passaged for further expansion. LE spheres were harvested using a cell scraper, washed with PBS, and dissociated into a single cell suspension by Accutase treatment for 10 min at 37 C followed by vigorous pipetting. Single cells were seeded on Matrigel, incubated at 37 C overnight, and subjected to 3D culture described above. After 2-4 rounds of expansion, LE cells (nearly 100% were EpCAM+) were stocked after obtaining single cell suspension. For transformation, LE cells were seeded onto tissue culture plates without Matrigel for 2D-culture, and infected with ecotropic retrovirus using standard procedures. Infected cells were selected in puromycin and used for subsequent studies. Proliferation Assay In Vitro Real-time cell proliferation assay was performed using an IncuCyte cell analyzer (Essen Bioscience, Ann Arbor, MI). Transplantation Experiments In allograft experiments, 1 x 106 of MEF-KRASG12V or LE-KRASG12V cells were injected s.c. into the dorsal flank of nude mice. MEFEGFRex19del and LE-EGFRex19del cells were injected as 1:1 mixtures with Matrigel at 2 x 106 cells per injection site. Tumor length and width were measured by caliper and volume calculated based on the standard formula (length x width2)/2. Xenografts using human SCLC and NSCLC cell lines were generated by s.c. inoculation of cells at 1 x 106 cells with Matrigel per site into NOG mice. PK Activity Assay PKase activity was measured using the Pyruvate Kinase Assay kit (BioVision, Milpitas, CA) where the generation of pyruvate were coupled with coloring of a probe through pyruvate oxidase. For MEFs, cells glucose-starved for 4 hr were refed with 1000 mg/L of glucose for 1 hr or left untreated, lysed, and assayed for PK activity without supplementation with fructose-1,6-bisphosphate (FBP) according to the manufacturer’s recommendation. For human lung cancer lines, cells were cultured 1 day in reduced glucose medium (1 mM glucose) and assayed for PK activity with or without extra FBP. Results shown in Figures 3B, 6C, and S6D were obtained using 1 mg protein in a reaction volume of 100 ml. Recombinant HA-tagged human PKM1 and PKM2 proteins were affinity-purified from 293T cells transiently transfected either with pCMV-HA-hPKM1 or pCMV-HA-hPKM2 using a HA-tagged Protein PURIFICATION KIT (MBL), according to the manufacturer’s recommendations. For assays using purified HA-PKM1 and HA-PKM2, 300 ng of each was diluted to 8 ml in TBS and then either 2 ml of 0.5 M FBP or H2O was added and the mixture incubated 10 min on ice. Then, to start the reaction 5 ml enzyme was added to 95 ml of reaction mixture. We reduced substrate (phosphoenol pyruvate (PEP)) levels in reactions to one-third of that recommended in the manufacturer’s protocol in order to detect allosteric regulation of PKM2. For kinetic parameter analyses, we used a lactate dehydrogenase (LDH)-coupled assay for PK activity, where pyruvate generation by PKase is coupled to NADH depletion through LDH activity (Christofk et al., 2008). Either 700 ng of HA-PKM1 or HA-PKM2 or a mixture of 350 ng each in 5 ml PBS was added to 1 ml H2O or 0.5 M FBP and incubated on ice for 10 min. Reactions were started by addition of 95 ml reaction mixture containing 50 mM Tris, pH7.5, 100 mM KCl, 10 mM MgCl2, 200 mM NADH, 8 units LDH, and various concentrations of ADP and PEP. When the assay were performed at increasing concentrations of PEP, ADP was fixed to 2 mM. When the assay were performed at increasing concentrations of ADP, PEP was fixed to 2 mM. The reduction in absorbance at 340 nm due to NADH oxidation was measured as an indicator of PK activity using a Sunrise plate-reader (TECAN). Data were fitted to a Michaelis-Menten equation using GraphPad prism software. Enzymatic Quantification of Metabolites To obtain spent medium, 1 x 106 cells were seeded in 10 cm dishes, and the next day cells were replenished with fresh medium and cultured 24 hr longer for lactate-release assays. Cells were then collected and lysed. Cellular protein levels were determined to e5 Cancer Cell 33, 355–367.e1–e7, March 12, 2018

normalize metabolite levels in medium. Lactate levels in conditioned medium were measured using a Lactate assay kit-II from BioVision. Cellular amino acid levels were measured using a L-Amino Acids Quantification kit from BioVision. Metabolite Measurement by Mass Spectrometry After removal of medium, cells were washed with PBS and then with 5% (w/v) mannitol solution. Metabolites were extracted from cells and spent medium with methanol containing internal standards (20 mM each of methionine sulfone, 2-(N-morpholino)ethanesulfonic acid (MES) and D-camphor-10-sulfonic acid (CSA)) for 10 min followed by chloroform extraction. The resulting aqueous phase was centrifugally filtered through a 5-kDa cut-off filter to remove proteins. The filtrate was evaporated, and then dissolved in Milli-Q water containing reference compounds (200 mM each of 3-aminopyrrolidine and trimesate) immediately prior to MS analyses. Measurements for cellular metabolites were performed using a capillary electrophoresis (CE)-mass spectrometry (MS) system, as described (Satoh et al., 2017; Yamamoto et al., 2014). Metabolite measurements for medium were carried out by using ion chromatography-MS and LC/MS, as described (Miyajima et al., 2017). Metabolite extraction and MS analyses of 13C-labbeled tumors were conducted at Human Metabolome Technologies, Inc. (Tsuruoka, Japan) according to F-scope service package. Briefly, approximately 50 mg of frozen tissue was plunged into 1,500 mL of 50% acetonitrile/Milli-Q water containing internal standards. The tissue was homogenized twice at 1,500 rpm for 2 min using a tissue homogenizer (Micro Smash MS100R, Tomy Digital Biology, Tokyo, Japan), and then the homogenate was centrifuged at 2,300 3g and 4 C for 5 min. Subsequently, 800 mL of upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The filtrate was evaporated and re-suspended in 50 mL of Milli-Q water for CE-MS analysis. Tracer Labeling Experiments In Vitro Cells were seeded at 1 x 106 cells per 10 cm dish at day 0, and fresh medium was added the next day (day 1). At day 2, medium was replaced with glucose-free DMEM (Wako, Osaka, Japan) supplemented with 10% FCS (dialyzed against PBS) and 1000 mg/L of [U-13C] glucose. Cells were labeled with [U-13C] glucose for additional 0.5-4 hr, washed, fixed with methanol and subjected to metabolite extraction as above. Alternatively, cells were labeled with 2 mM [U-13C] glutamine for 2-6 hr. Tracer Labeling Experiments In Vivo Nude mice bearing allograft tumors (0.5-1 cm diameter) in the flank were fasted for 16 hr and then anesthetized by inhalation with 0.2% isoflurane consistently at close to 9 AM to minimize potential circadian effect. After 5-10 min, [U-13C] glucose was intraperitoneally injected at 2 g/kg, and then mice were left on a heating mat for an additional 0.5-1 hr under anesthesia. Mice were euthanized at the end of the labeling period, and tumors were harvested, rinsed with cold PBS and snap frozen in liquid nitrogen. Measurement of Oxygen Consumption Rates OCRs of LEs and MEFs were measured using a Seahorse XF96 flux analyzer (Agilent, Santa Clara, CA). For the assay cell medium was DMEM (unbuffered) containing 4500 mg/L glucose, 0.29 mg/ml glutamine and no pyruvate. After the 3 measurements of basal OCR, rotenone and antimycin A1 were added at 3 mM each to inhibit electron transport chain. Rotenone/antimycin-sensitive OCR was considered mitochondrial OCR. After analysis, cells were fixed with trichloroacetic acid and stained with sulphorhodamine B (Sigma), and OCR was normalized to number of cells, as described (Kaplon et al., 2013). Mitochondria and ROS Assays Cells were stained with MitoTracker DeepRed, MitoTracker Green, MitoSOX Red, CellROX Green or CellROX DeepRed (all from Thermo Fisher Scientific, Waltham, MA) according to instructions and analyzed using a Synergy H1 Hybrid Multi-Mode Monochromator plate reader (BioTek, Winooski, VT). Cells were then fixed in paraformaldehyde and stained with DAPI. DAPI signal values served as an internal control. Generation of Atg7-Knockout Cells Atg7 guide RNA designed using a CRISPR Design tool (http://crispr.mit.edu/) was subcloned into pX330-U6-Chimeric_BB-CBhhSpCas9 (a gift of Dr F. Zhang, Addgene #42230 (Cong et al., 2013)), a human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. MEFs were co-transfected with the pX330 and pEGFP-C1 (Clontech Laboratories Inc., Mountain View, CA) vectors and cultured 2 days. GFP-positive cells were then sorted and expanded. Loss of Atg7 was confirmed by heteroduplex mobility assay followed by immunoblot with anti-ATG7 antibody as described previously (Muona et al., 2016). Autophagic Probe Assay The GFP-LC3-RFP construct (Kaizuka et al., 2016), provided by Dr N. Mizushima, was introduced into MEFs using a retrovirus generated by PLAT-E cells. MEFs expressing GFP-LC3-RFP were seeded at 1 x 104 cells/well in 96-well plates. The next day, cells were treated with 50 nM BEZ235 for 1 more day to activate autophagy. GFP and RFP signals were quantified using Synergy H1 as described previously (Kaizuka et al., 2016).

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Transfection Experiments HeLa cells were transiently transfected with pCMV-HA-PKM1, pCMV-Myc-PKM1 or pCMV-HA-PKM2 plasmids, separately or in combination, using LipofectAMINE2000, following the manufacturer’s recommendations. For co-immunoprecipitation experiments, cells were lysed by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mN NaCl, 5 mM EDTA and 1% Brij45. Immunoprecipitations were done using a standard procedure using anti-myc #9e10 mAb and ProteinG-Sepharose. For BN-PAGE analyses, cells were lysed in 1 x BN buffer containing protease inhibitors. Blue-Native PAGE Analysis 0.3 mg of HA-PKM1 and/or HA-PKM2 in PBS was incubated with or without 0.1 M FBP on ice for 10 min (in a volume of 10 ml). Then, 2.5 ml of 4x Blue Native (BN)-PAGE sample buffer (Thermo Fisher Scientific) was added to samples, which were subjected to BN-PAGE. Gels were then stained with Coomassie Brilliant Blue G250. Alternatively, for immuno-blotting, halfway through the electrophoresis procedure, the cathode buffer was changed to G250-free one to remove excess G250 dye before transferring proteins to PVDF membranes. After electroblotting, the membrane was first soaked in Denaturing buffer (50 mM Tris-HCl, pH 7.4, 2% SDS, 0.8% 2-mercaptoethanol) at 50 C for 10 min (3 times) to retrieve antigens and then subjected to immunoblotting according to standard protocols. For ESC experiments, WT and heterozygous PkmM1/+ ESCs were cultured in gelatin-coated dishes without feeder cells. Recovered cells were directly lysed in 1x BN-PAGE buffer and subjected to BN-PAGE. SCLC Cells Expressing Exogenous PKM1 or PKM2 Cells were transduced with mSlc7a1 (an ecotropic virus receptor) by lentiviral vector and selected with blasticidin-S. Lentivirus was packaged in 293 cells by transfection with plasmid pLenti6-UbC-mSlc7a1 (a gift of Dr S. Yamanaka (Takahashi et al., 2007)), psPAX2 and pMD2.G (gifts of Dr D. Trono, Addgene #12260, 12259) using Fugene6. Resulting cells expressing mSlc7a1 were infected with ecotropic retrovirus expressing mouse PKM1 or PKM2 cDNA (each Flag-tagged) and selected in hygromycin. Retrovirus was packaged in PLAT-E cells by transfection of pLHCX-Flag-mPKM1 or pLHCX-Flag-mPKM2 (gifts of Drs L. Cantley and MG. VanderHeiden, Addgene #44239, 44240 (Christofk et al., 2008)). Cells were then infected with lentivirus expressing shRNA against human PKM that targets both PKM1 and PKM2. shRNA-virus was produced using pLKO-shPKM2_4 (a gift of Dr D. Anastasiou, Addgene #42516 (Anastasiou et al., 2011) ) as described above. QUANTIFICATION AND STATISTICAL ANALYSIS No statistical methods were used to predetermine sample size. Experiments were not randomized nor were investigators blinded to allocation during experiments and outcome assessment. In experiments shown in Figure 1F, samples were excluded from final analyses if neoplastic lesions were not observed in lung, an outcome likely due to adenovirus-injection failure. Animal experiments shown in Figure 1 were each performed once. qRT-PCR and MS spec analyses shown in Figures 1, 2, 5, S2, and S5, in vivo tracer experiments in Figure S3 were each performed once at 2 or 3 measurement duplicates. All other experiments were performed at least twice. We used Student’s t-test (2-tailed) and a one-way ANOVA followed by a post hoc test when comparing two groups and multiple groups, respectively. A p value of <0.05 was considered significant. Data are presented as mean with the range (Figure 3B) or SEM (all others). DATA AND SOFTWARE AVAILABILITY Metabolomics data has been deposited in the Mendeley Data as noted in the Key Resources Table (https://doi.org/10.17632/ 9pvjp45zgn.1). All other data relevant to this study are available by request from the corresponding author.

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