Johanna Chiche

Julie Reverso-Meinietti

Annabelle Mouchotte

Camila Rubio-Patino

Rana Mhaidly

Elodie Villa

Jozef P Bossowski

Emma Proics

Manuel Grima-Reyes

Agnes Paquet

Camila Rubio-Patiño

Agnès Paquet

Konstantina Fragaki

Sandrine Marchetti

Josette Briere

Damien Ambrosetti

Jean-François Michiels

Thierry Jo Molina

Christiane Copie-Bergman

Jacqueline Lehmann-Che

Isabelle Peyrottes

Frederic Peyrade

Eric De Kerviler

Bruno Taillan

Georges Garnier

Els Verhoeyen

Véronique Paquis-Flucklinger

Laetitia Shintu

Vincent Delwail

Celine Delpech- Debiais

Richard Delarue

André Bosly

Tony Petrella

Gabriel Brisou

Bertrand Nadel

Pascal Barbry

Nicolas Mounier

M.D Catherine Thieblemont
email: catherine.thieblemont@sls.aphp.fr

Ph.D Jean-Ehrland Ricci
email: ricci@unice.fr

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors

Keywords: DLCBL, R-CHOP, metabolism, OxPhos, glycolysis, mTOR, predictive marker, Lasparaginase

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous group of tumors and the most common type of lymphoid malignancies [START_REF] Swerdlow | The 2016 revision of the World Health Organization classification of lymphoid neoplasms[END_REF]. DLBCLs represent highly metabolically active tumors previously treated with a polychemotherapy CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin® and prednisone) [START_REF] Fisher | Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin's lymphoma[END_REF]. Since 1998, immunotherapy anti-CD20 (Rituximab) combined with CHOP (referred as R-CHOP) became the standard treatment for this disease, as it significantly improved the survival of patients [START_REF] Coiffier | CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma[END_REF]. However, approximately 40% of patients with DLBCL still experience therapeutic failure upon R-CHOP [START_REF] Sehn | Diffuse large B-cell lymphoma: optimizing outcome in the context of clinical and biologic heterogeneity[END_REF]. DLBCLs molecular heterogeneity is considered a major factor influencing the response to R-CHOP therapy [START_REF] Lenz | Aggressive lymphomas[END_REF][START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. The cell-of-origin (COO) classification delineates DLBCL subsets into distinct transcriptional profiles: the Germinal Center Bcell (GCB)-, the Activated B-cell (ABC)-and the Unclassified-DLBCL subtypes [START_REF] Alizadeh | Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling[END_REF]. Unlike GCB-DLBCLs, ABC-DLBCLs are associated with a poor outcome upon R-CHOP treatment [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. Comparison of other gene signatures highlighted three other DLBCL clusters that are recapitulated in the consensus cluster classification (CCC): i) the B cell receptor (BCR)/proliferation (BCR-DLBCL) up-regulates genes encoding BCR signaling components; ii) the oxidative phosphorylation (OxPhos) cluster (OxPhos-DLBCL) is enriched in genes involved in electron transport chain (ETC) complexes, OxPhos metabolism and in other mitochondrial functions and iii) the host response (HR) cluster is characterized by a host inflammatory infiltrate [START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF]. Importantly, there is no link between the COO and the CCC classifications [START_REF] Caro | Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma[END_REF][START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF]. Further characterization demonstrated that OxPhos-DLBCL cell lines lack the B cell receptor and are insensitive to inhibition of the BCR signaling pathway [START_REF] Chen | SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma[END_REF]. In contrast, BCR-DLBCL cell lines express a functional BCR and primarily rely on glycolysis to produce energy [START_REF] Caro | Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma[END_REF][START_REF] Chen | SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma[END_REF]. Altogether those studies have identified, for the first time, the metabolic heterogeneity within the same tumor entity [START_REF] Caro | Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma[END_REF][START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF][START_REF] Norberg | Differential contribution of the mitochondrial translation pathway to the survival of diffuse large B-cell lymphoma subsets[END_REF]. However, whether distinct metabolic fingerprints influence DLBCL response to R-CHOP remains unknown. Moreover, it is unclear whether targeting DLBCL metabolic specificity might be a valuable therapeutic approach in the clinic, in particular for R-CHOP low responder patients. In this study, we addressed two unmet clinical needs: can we identify, at the diagnosis, which DLCBL patients is likely to be a low responder to R-CHOP? and can we foretell if those patients will be likely to respond to specific metabolic inhibitors according to their DLBCL's metabolic status?

Using unbiased analysis, we identified the glyceraldehyde-3-phosphate dehydrogenase, GAPDH, as the only glycolytic enzyme able to predict overall survival of patients with DLBCL treated with R-CHOP. Moreover, we established in vivo as well as in the clinic, that GAPDH holds potential as a marker of DLBCL metabolic status and of patients's response to specific metabolic inhibitors.

Results

GAPDH expression predicts overall survival of patients with DLBCL treated with R-CHOP and DLBCL metabolic status.

Unbiased analysis of expression profile data set from 233 newly diagnosed DLBCLs [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF] allowed us to identify 587 probesets (p<0.05), corresponding to 524 unique genes associated with differences in overall survival (OS) upon R-CHOP treatment (Table S1,Fig S1A). Analysis of molecular functions associated with OS predicting genes, identified classical pathways involved in resistance to chemotherapies (cell survival, proliferation, DNA repair…) and highlighted the mitochondrial energetic function (Table S1 and Fig S1B). In contrast, only one gene involved in glycolysis, the glyceraldehyde-3-phosphate dehydrogenase (gapdh) predicts a favorable outcome when highly expressed (3 independent probesets: HR= 0.53, 0.53 and 0.54 and corresponding p value= 0.01573, 0.01771 and 0.01929; Table S1). Of note, we also observed that lactate dehydrogenase b subunit (ldhb) is significantly associated with a poor outcome (Table S1). Though, LDH-B converts lactate to pyruvate, a source for OxPhos metabolism or for gluconeogenesis [START_REF] Doherty | Targeting lactate metabolism for cancer therapeutics[END_REF], we focused our attention on gapdh.

When subgrouping DLBCLs according to gapdh mRNA levels (high or low,Fig 1A), patients with DLBCL-gapdh low have a lower OS upon R-CHOP than those with DLBCL-gapdh high (Fig 1B). To further determine whether GAPDH protein expression predicts differences in OS upon R-CHOP, we set up an automated immuno-histochemical (IHC) staining of GAPDH and a scoring system to quantify its expression in 43 newly diagnosed DLBCL (training cohort, Table S2). As for gapdh mRNA (Fig 1B), DLBCLs expressing low levels of GAPDH protein (GAPDH low , score ≤ 150) are associated with a poorer OS upon R-CHOP than DLBCLs expressing high levels of GAPDH (GAPDH high , score > 150) (Figs 1D and 1E, see also STAR methods for determination of GAPDH IHC score and cutoff of 150). This finding was confirmed in a larger cohort of 294 de novo DLBCLs treated with R-CHOP (validation cohort, Table S2) [START_REF] Delarue | Dose-dense rituximab-CHOP compared with standard rituximab-CHOP in elderly patients with diffuse large B-cell lymphoma (the LNH03-6B study): a randomised phase 3 trial[END_REF] by staining and scoring GAPDH from paraffin-embedded tissue microarray (TMA) using the previously established IHC score cutoff (Figs 1F and 1G).

Interestingly, GAPDH expression is not associated with clinical factors such as age-adjusted International Prognostic Index (aaIPI), COO or Hans classification [START_REF] Hans | Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray[END_REF], expression of bad prognosis markers, double hit (MYC/BCL2) or triple hit (MYC/BCL2/BCL6) translocations previously validated in DLBCLs [START_REF] Horn | MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma[END_REF][START_REF] Johnson | Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone[END_REF] (Table 1). Of note, there is no difference in OS noted on the basis of BCL6, or MYC or MYC-BCL2 expressions in the validation cohort. However, BCL2 expression significantly impacts the OS [START_REF] Petrella | BCL2 expression but not MYC and BCL2 coexpression predicts survival in elderly patients with diffuse large B-cell lymphoma independently of cell of origin in the phase 3 LNH03-6B trial[END_REF]. Multivariate analysis showed that GAPDH high expression remained a significant marker predicting improved OS (HR=0.603, p=0.0371), independently of BCL2 (HR=1.686, p=0.0213). When compared to the aaIPI, multivariate analysis showed that GAPDH expression also remained a significant marker predicting improved OS in the training and validation cohorts (Table S3).

Altogether, these results demonstrate that the predictive value of GAPDH expression levels is independent of known biomarkers/factors of poor prognosis.

According to the CCC classification, we observed that 69% of DLBCL-gapdh low (n=49/71) are OxPhos-DLBCLs, while 61% of DLBCL-gapdh high (n=51/82) belong to the BCR/proliferation cluster associated with glycolysis (two-sided p=0.00019; analysis from (Lenz et al., 2008) cohort). OxPhos-DLBCLs significantly express lower levels of gapdh mRNA than . In addition, we did not observe association between GAPDH expression levels and specific COO subtypes in the training and validation cohorts (Table S2), consistent with the absence of correlation between the CCC and the COO classification [START_REF] Caro | Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma[END_REF][START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF]. Moreover, patients with BCR-or OxPhos-DLBCL display the same outcome upon CHOP treatment [START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF]. Accordingly, distinct gapdh mRNA levels display similar OS when DLBCL are treated with the polychemotherapy CHOP only (Fig S1C).

Arguments from the literature suggest that OxPhos-DLBCL-might be less sensitive than BCR-DLBCL to Rituximab due to the lack of Syk (Spleen Tyrosine Kinase)-dependent BCR signaling [START_REF] Kheirallah | Rituximab inhibits B-cell receptor signaling[END_REF]. Compared to BCR-DLBCL cell lines (SU-DHL-4 and SU-DHL-6), i.e glycolytic lymphoma cells expressing a functional BCR (IgM or IgG) [START_REF] Chen | SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma[END_REF], OxPhos-DLBCL cell lines (Toledo and Karpas 422) express lower levels of GAPDH, secrete less lactate, express neither IgM nor IgG and are less sensitive to Rituximab-induced death (Figs S2A to S2D). Of note, GAPDH expression (mRNA and protein) is not influenced upon BCR activation with anti-IgM in SU- . In another approach, we investigated the energetic metabolic status of malignant B cells harvested from fresh micro-biopsies of primary DLBCL tumors, according to the level of GAPDH. DLBCL-GAPDH low cells produce less glycolytic ATP than DLBCL-GAPDH high cells and there is a significant correlation between GAPDH expression level, and the percentage ATP generated from glycolysis (Fig 1I).

We concluded that DLBCL-GAPDH low are low responders to R-CHOP treatment and mainly rely on OxPhos metabolism to fulfill their energetic demand. Using the pre-clinical Eµ-Myc mouse model that spontaneously develop Non-Hodgkin (NH) B-cell lymphomas [START_REF] Adams | The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice[END_REF], we wondered whether GAPDH expression levels could influence the metabolic status of B lymphomas and the response to disruptors of mitochondrial metabolism in vitro and in vivo. We previously characterized the expression of GAPDH in Eµ-Myc lymphomas and classified them according to the level of GAPDH (mRNA and protein) (GAPDH low or GAPDH high ), while the expression of the other glycolytic enzymes is similar between the two groups ( [START_REF] Chiche | GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin's B lymphomas via NF-kappaB-dependent induction of HIF-1alpha[END_REF] and Figs S3A to S3C). Of note, we did not observe any link between GAPDH and LDH (total or LDH-B) expression (Fig S3A). While the exact nature of GAPDH regulation remains to be found, gapdh mRNA was more stable in GAPDH high than in GAPDH low cells ( In vitro, the mitochondrial complex I inhibitor, phenformin [START_REF] Wheaton | Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis[END_REF] reduces baseline O2 consumption driven by complex I and induces cytotoxic responses in Eµ-Myc-GAPDH low cells but not in Eµ-Myc-GAPDH high cells (Figs 2E and S3G to S3I). Importantly, gapdh-silencing in Eµ-Myc-GAPDH high cells decreases lactate production and increases cell sensitivity to phenformin-induced death (Figs S4A to S4D). In vivo, phenformin treatment increases the survival of Eµ-Myc-GAPDH low lymphoma-bearing mice, but not that of Eµ-Myc-GAPDH high lymphoma-bearing mice (Figs 2 F and 2G), which demonstrates the causal correlation between low expression of GAPDH and phenformin sensitivity. We confirmed that phenformin was indeed active at the dose administrated in vivo (Figs

Mouse primary B lymphomas expressing low levels of GAPDH

S3J-and S3K).

Overall, Eµ-Myc-GAPDH low lymphomas are sensitive to the inhibition of mitochondrial metabolism, which represents a potential therapeutic strategy for patients with DLCBL-GAPDH low that are low responders to R-CHOP treatment.

Active mTORC1 signaling in GAPDH low lymphomas is implicated in OxPhos metabolism.

GAPDH was shown to modulate mTOR signaling through its binding to Rheb [START_REF] Lee | Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenasemediated regulation of Rheb[END_REF]. We therefore speculated that mTORC1 could be active in GAPDH low B lymphomas. Indeed, we observed a high mTORC1 activity in Eµ-Myc-GAPDH low lymphomas, as shown by p70-S6K phosphorylation on threonine 389 (Fig 3A). This was confirmed in human DLBCL biopsies, for which 57% of DLBCL-GAPDH low stained positive for p70-S6K phosphorylation (indicating high mTORC1 activity), while 91% of DLBCL-GAPDH high do not express this form (indicating low mTORC1 activity) (Fig

3B).

Inhibition of mTORC1 signaling with rapamycin in Eµ-Myc-GAPDH low cells, decreases basal O2 consumption rate, maximal respiratory capacities and ATP-coupled OCR along with an increase in glycolytic ATP (Figs 3 C to 3G). Of note, the total amount of intracellular ATP was not modulated upon rapamycin treatment (Fig S5A). More specifically, rapamycin decreases O2 consumption driven by complex I but not complex II (Figs S5B and S5C), due to the inhibition of complex I activity but not of complex II activity (Fig S5D). The exact reason of such effect remains to be determined. Similar results were obtained using an independent Eµ-Myc-GAPDH low lymphoma (Figs S5E to S5G).

As previously reported in several cancer cell lines [START_REF] Fruman | PI3K and cancer: lessons, challenges and opportunities[END_REF] These results suggest that mTORC1 signaling is involved in the control of GAPDH low B lymphoma metabolism.

B lymphomas expressing low levels of GAPDH rely on glutamine metabolism and are sensitive to the hydrolysis of extracellular glutamine and asparagine with E-Coli L-asparaginase.

mTORC1 is a central sensor of amino acids, including Gln [START_REF] Duran | Glutaminolysis activates Rag-mTORC1 signaling[END_REF]. Eµ-Myc-GAPDH low lymphomas display active mTORC1 signaling (Fig 3A) and consume more Gln than the Eµ-Myc-GAPDH high cells (Fig 4A) due to a specific increase in Gln transport rates (Figs 4B,S6A and S6B).

We next profiled metabolic differences between Eµ-Myc-GAPDH low and Eµ-Myc-GAPDH high lymphoma cells using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics with the intent to further characterize the intracellular fate of Gln (Fig 4C). The relative intracellular levels of metabolites involved in glycolysis (G6P, G3P, lactate) and in the non-oxidative arm of the pentose phosphate pathway (PPP) (Sedoheptulose-7-phosphate) are significantly decreased in Eµ-Myc-GAPDH low cells (Fig 4C). Glutamine is metabolized through glutaminolysis to produce glutamate, αketoglutarate, the latter being used to replenish the TCA cycle. In favor of an active glutaminolysis, glutamate and α-ketoglutarate levels are higher in Eµ-Myc-GAPDH low cells (Fig 4C ), consistent with an increase expression of the glutaminase (GLS), the mitochondrial enzyme catalyzing the conversion of glutamine into glutamate and ammonia (Fig 4D). While fewer metabolites could be detected by 1 H HRMAS NMR spectroscopy, a significant increase in intracellular glutamate levels was also confirmed in Eµ-Myc-GAPDH low cells (Figs S6C andS6D 4I). Of note, E-Coli L-ase treatment inhibited mTORC1 signaling in vivo (Figs S6I and S6J).

Specific overexpression of GAPDH reduces mTORC1 signaling and B lymphomas sensitivity to phenformin and L-asparaginase in vivo.

To further support our conclusions, we overexpressed GAPDH in Eµ-Myc-GAPDH low cells (Fig 5A ). Together we confirmed that GAPDH expression influences the metabolic state and the sensitivity of B lymphomas to phenformin or E-Coli L-asparaginase.

Treatment of DLBCL-GAPDH low with specific metabolic inhibitors demonstrates a significant benefit for patients.

We demonstrated that GAPDH low B lymphomas preferentially use OxPhos metabolism to produce energy and are sensitive to cell death induced by phenformin or E-Coli L-asparaginase. To ultimately validate our findings, we designed a clinical therapeutic intervention (called KTM), to interfere with DLBCL-GAPDH low metabolism (Fig 6A). KTM therapy consists in 4 weeks-cycles of treatment including E-Coli L-asparaginase (K, Kidrolase), mTOR inhibitor Temsirolimus (T, Torisel) and Metformin (Fig 6B). The first two weeks, patients receive E-Coli L-asaparaginase on days 1, 3, 5, 7, 9, 11, 13 and Temsirolimus on days 1, 7 and 14. This combination was not administered longer than 14 days since E-Coli L-asparaginase is not well tolerated in adults [START_REF] Boissel | Best Practices in Adolescent and Young Adult Patients with Acute Lymphoblastic Leukemia: A Focus on Asparaginase[END_REF]. Instead, the last two weeks of each KTM cycle, patients received metformin to sustain a therapeutic pressure on mitochondrial metabolism (Fig 6B). It is important to consider that prior to KTM, patients were refractory to all R-based therapies and only supportive care was proposed.

Discussion:

Several metabolic inhibitors were developed with the intent to kill or to sensitize cancer cells to chemotherapies [START_REF] Galluzzi | Metabolic targets for cancer therapy[END_REF]. So far, more than 100 clinical trials using metabolic inhibitors are ongoing in the field of cancer [START_REF] Meynet | Caloric restriction and cancer: molecular mechanisms and clinical implications[END_REF]. Unfortunately, to date, most of those trials have failed to improve the outcome of patients [START_REF] Kordes | Metformin in patients with advanced pancreatic cancer: a double-blind, randomised, placebo-controlled phase 2 trial[END_REF]. This may not be linked to the inefficacy of the compound to inhibit its target but rather to a subset of tumors with a distinct metabolic dependence from the targeted metabolic pathway. Altogether, our study highlighted that tumor heterogeneity within the same tumor entity has to be taken into consideration for ongoing clinical trials using metabolic inhibitors.

While, more than 150'000 claimed biomarkers have been described in the literature, less than 100 are routinely used in the clinic [START_REF] Poste | Bring on the biomarkers[END_REF] and we still lack a marker of the tumor metabolic status.

Here, we identified GAPDH, as a clinically compatible biomarker of i) the conventional R-CHOP treatment and of ii) the metabolic status of DLBCLs, which help to further predict the efficacy of mitochondrial metabolism disruptors in the context of DLCBL-GAPDH low that are associated with a poor outcome of patients treated with R-CHOP.

GAPDH is a multifunctional protein harboring glycolytic and non-glycolytic functions [START_REF] Colell | Novel roles for GAPDH in cell death and carcinogenesis[END_REF]. It is highly regulated at the transcriptional, translational and post-translational levels.

Endogenous gapdh mRNA and protein are highly abundant in all mammalian cells and increased expression levels of gapdh by 30% or more, significantly impacts on cell survival and growth [START_REF] Colell | Novel roles for GAPDH in cell death and carcinogenesis[END_REF], particularly in B lymphomas [START_REF] Chiche | GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin's B lymphomas via NF-kappaB-dependent induction of HIF-1alpha[END_REF]. We observed that human DLBCL expressed heterogeneous levels of gapdh mRNA and protein defining patients with DLBCL-GAPDH low or DLBCL-GAPDH high . As expected, when comparing healthy tissues (GCB cells from non-cancerous reactive lymph nodes) to cancer samples, DLBCL-GAPDH low express significantly higher levels of GAPDH (Fig 1D). Moreover, when compared to DLBCL-GAPDH low , DLBCL-GAPDH high express approximatively two times more GAPDH, which also highlights GAPDH as a marker of tumor heterogeneity within the same tumor entity. Interestingly, gapdh mRNA stability was higher in Eµ-Myc-GAPDH high cells (Fig S3D). As GAPDH is an RNA binding protein [START_REF] Colell | Novel roles for GAPDH in cell death and carcinogenesis[END_REF], we could speculate that it could bind and stabilize its own mRNA. However, the exact nature of gapdh regulation in DLBCL is still unclear and will be investigated in further studies. Mechanistically, we observed a central role of GAPDH-dependent regulation of mTORC1 signaling in the control of DLBCL metabolic status. It was previously reported that gapdh-silenced HEK293 cells increase mTORC1 signaling by preventing GAPDH-Rheb binding [START_REF] Lee | Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenasemediated regulation of Rheb[END_REF]. Using the Eµ-Myc model and newly diagnosed DLBCL samples we also observed an increase in mTORC1 signaling, glutamine uptake, glutaminolysis and sensitivity to L-asparaginase-induced death in GAPDH low B lymphomas (Fig 3 and4). Altogether our findings are consistent with the role of functional mTORC1 on glutamine uptake [START_REF] Csibi | The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-dependent control of c-Myc translation[END_REF] and the feedback role of glutaminolysis on the activation of Rag-mTORC1 signaling [START_REF] Duran | Glutaminolysis activates Rag-mTORC1 signaling[END_REF]. How GAPDH-dependent control of mTORC1 regulates metabolism? mTORC1 is a known-regulator of HIF-1α and c-Myc translation, which in turn induce the expression of genes involved in glucose uptake and glycolysis [START_REF] Gordan | HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation[END_REF]. In primary Eµ-Myc lymphomas, we did not observe differences in glucose transporters expression (Fig S3E) according to GAPDH expression levels, despite differences in mTORC1 signaling. In contrast, inhibition of mTORC1 reduces O2 consumption of Eµ-Myc GAPDH low cells, in agreement with previous studies [START_REF] Cunningham | mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex[END_REF][START_REF] Morita | mTORC1 controls mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation[END_REF]. It seems therefore possible that Eµ-Myc lymphomas and human DLBCLs do not follow the classical Warburg scheme in which mTORC1 activates glycolysis. Instead, mTORC1 seems implicated in the regulation of OxPhos metabolism in this biological model (Fig 3). This notion is further supported by the increase in glutamine uptake, along with an increase in the relative intracellular levels of glutamate and αketoglutarate and L-asparaginase efficacy on reduced mTORC1 signaling in vitro and in vivo (Willems et al., 2013) (Figs 4 and S6).

Since IHC detection of GAPDH seemed to help for the determination of the tumor metabolic status, we selected four patients with Myc + -DLBCL-GAPDH low . All patients were refractory to R-based therapies. In the face of a therapeutic dead end, we ultimately target DLBCLs-GAPDH low metabolism with already available (FDA-approved) metabolic disruptors (KTM therapy) (Fig 6). Based on the observed cytostatic effect of Temsirolimus in vitro and in vivo (Figs 3I and S5H), we decided still to administer this mTOR inhibitor either with L-asparaginase (D1, D7 of a KTR cycle) or with Metformin (D14 of a KTR cycle) to maximize the potential beneficial effect of the co-treatment.

However, knowing that rapalogs alone or in combination with R-CHOP did not provide significant benefits in randomized phase II and phase III trials for patients with NH B-cell lymphomas [START_REF] Ricci | Metabolic Reprogramming of Non-Hodgkin's B-Cell Lymphomas and Potential Therapeutic Strategies[END_REF], it is unlikely that the rapid and complete responses obtained after one week of KTM treatment can be attributed to Temsirolimus. According to the beneficial effect observed in Eµ-Myc-GAPDH low lymphomas upon E-Coli L-ase (Kidrolase) and to the early reduction of the tumor burden observed from D7 in patient #1 (Fig 6C ), it is likely that most of the treatment efficacy can be attributed to the L-ase activity. Unfortunately, in the clinic, E-Coli L-ase cannot be administrated longer than two weeks in a row in adults, as it may provoke major toxicities [START_REF] Boissel | Best Practices in Adolescent and Young Adult Patients with Acute Lymphoblastic Leukemia: A Focus on Asparaginase[END_REF]. We therefore sustained inhibition of mitochondrial energetics with metformin and mTOR inhibitor for the last two weeks of KTM cycle of treatment (Fig 6B). Despite the drastic reduction of the tumor mass, all patients finally relapsed, which very likely reflect the metabolic flexibility and adaptation of the remaining malignant B cells during or after KTM therapy.

Limitations of study

A major limitation in the field of DLBCL is a lack of robust pre-clinical models (cell lines or in vivo mouse models) that recapitulate the major aspects of the human disease. As human cancer cell lines were isolated decades ago and since grown in high glucose, high oxygen media, it is possible that their metabolism do not reflect the metabolic status of the initial tumor cells. We therefore decided to characterize several independent murine primary NH B cell lymphomas harvested from the Eµ-Myc mice model (Figs 2 to 5). While this model represents highly aggressive tumors (Ki67 100%) and is largely used as a robust model to test new therapeutic options for NH B lymphomas, they lack key features of human DLBCL such as marker of mature B cells. We therefore validated our findings in humans, using malignant B cells immediately isolated from "fresh" DLBCL micro-biopsies and ultimately by treating a few patients suffering from refractory disease with specific metabolic inhibitors (Figs 1, 3 and6). Another limitation of our work is the low number of patients included in the KTM therapy. However, our study established the proof-of-principle that patients with DLBCL-GAPDH low are low responders to immuno-chemotherapy R-CHOP but are potential candidates for KTM therapy. Finally, while GAPDH levels represent the first clinically compatible marker of DLBCL metabolic heterogeneity, it remains to be determined if this marker can be extended to other hematological malignances and to solid tumors.

Declaration of Interests,

CT is on the scientific board for Jazz Pharmaceuticals; Celgene; Roche, AbbVie; and Gilead.

The remaining authors declare no conflicts of interest. A. Distribution of gapdh expression values (log2 mRNA intensity) in newly diagnosed DLBCLs characterized as DLBCLs-gapdh low (n=125) and DLBCLs-gapdh high (n=108) (analysis from [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. p value from Wilcoxon rank sum.

B.

Overall survival (OS) of R-CHOP-treated DLBCL patients, according to gapdh low (n=125) and gapdh high (n=108) mRNA levels in DLBCL biopsies at diagnosis (analysis from [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. p value from log-rank test. [START_REF] Monti | Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response[END_REF], analysis from ( [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. p value from Wilcoxon test.

I. Correlation between GAPDH expression levels scored by IHC and glycolytic ATP in malignant B

cells harvested from patients with newly diagnosed DLBCLs. Blue dots represent DLBCLs-GAPDH low (n=3) and red dots represent DLBCL-GAPDH high (n=8). R, Pearson correlation coefficient. p value from correlation test.

See also Tables S1, S2, S3 andFigures S1 andS2 A. Total cell extracts from mouse primary Eμ-Myc-GAPDH low cells (lymphoma #F) stably transduced with control (pMIG) or GAPDH-V5-encoding pMIG vectors were immunoblotted for the indicated proteins. Erk2 is used as a loading control.

B. Intracellular ATP levels produced in Eµ-Myc-GAPDH low cells stably overexpressing GAPDH-V5

or control vector (pMIG) were quantified using a luciferin/luciferase-based assay and results were expressed as relative luminescence units normalized by cell number (n=3 independent experiments).

C. Glycolytic ATP production was measured as the percentage of total ATP in cells presented in A.

Data are presented as means ± s.d (n=3 independent experiments). D. Lactate production was determined in the supernatant of mouse primary Eμ-Myc-GAPDH low cells (lymphoma #F) stably transduced with control (pMIG) or GAPDH-V5-encoding pMIG vectors, 24 hours after seeding. Data are presented as mean ± s.d (n=3 independent experiments). See also Table S4 related to Figure 6.

E. Baseline OCR of cells presented in

Tables with titles and legends, Table 1. GAPDH expression levels are not associated with other biological prognostic factors.

BCL2 ≥ 70% means that 70% (or more) of malignant B cells stained for BCL2 protein by IHC [START_REF] Green | Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone[END_REF][START_REF] Petrella | BCL2 expression but not MYC and BCL2 coexpression predicts survival in elderly patients with diffuse large B-cell lymphoma independently of cell of origin in the phase 3 LNH03-6B trial[END_REF]; BCL6 > 25% means up to 25% of malignant B cells stained for BCL6 by IHC [START_REF] Horn | MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma[END_REF]; MYC ≥ 40% [START_REF] Johnson | Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone[END_REF] means that 40% (or more) of malignant B cells stained for MYC protein by IHC. Double hit (MYC/BCL2) and triple hit (MYC/BCL2/BCL6) were obtained by Fluorescence In Situ Hybridization (FISH). p value from Fisher test. See also Tables S2 andS3, related to Table 1.

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jean-Ehrland Ricci (ricci@unice.fr).

Materials and Methods

Experimental model and subject details

Mice and in vivo studies using Eµ-Myc cells

C57BL/6 Eµ-Myc transgenic mice were purchased from the Jackson Laboratory (#002728) and housed in our local animal facility (C3M, Nice, France).

The lymphoma transfer of Eµ-Myc cells was performed into syngeneic, nontransgenic, 6-weeks-old C57BL/6JOlaHsd females (Envigo) by intravenous injection (iv) of 0.1 × 10 6 viable Eµ-Myc lymphoma cells per recipient mouse (in 150 µl of sterile PBS). Six days after cell injection, mice received treatments. 100 mg/kg/day of Phenformin (P7045, Sigma-Aldrich) or vehicle (H2O) (n=10 mice/group) were administrated by gavage (p.o) every day for three weeks. 2500 UI/kg/day of E-Coli L-asparaginase (Kidrolase, L-ase, Jazz Pharmaceutical) or vehicle (NaCl 0.9%) was injected intraperitoneally for five days the first week and three times per week the following weeks, for a total of three weeks of treatment (n=6 mice/group). 10 mg/kg of Temsirolimus (PZ0020, Sigma-Aldrich) or vehicle (NaCl 0.9%-5%Tween-5% PEG-200-1% EtOH) were intraperitoneally injected two times per week for three weeks (n=6 mice per group). Tumor onset was determined by inguinal lymph node palpation. Lymphoma-bearing animals were sacrificed by cervical dislocation. Phenformin-treated software.

DLBCL patients: Training & Validation cohorts

DLBCL tumors were required to be de novo and treated with Rituximab-CHOP (R-CHOP). All cases were diagnosed and reviewed by experienced hematopathologists and patient clinical information were obtained from the Hopital Saint Louis, Centre Hospitalier Princesse Grace and Centre Antoine Lacassagne institutions. DLBCLs were classified according to the WHO classification. Clinical data including gender, age, and age-adjusted International Prognosis Index (IPIaa) (scored (0-1, 2 and 3) based on i) the Ann Arbor stage (II-II vs III-IV), ii) the level of lactate dehydrogenase in the serum (LDH ≤ normal vs ≥ normal) and iii) the Performance status (PS 0-1 vs 2-4)) were collected in all cases and are summarized in Table S2. These cases were anonymized and processed in accordance with a protocol approved by scientific committee of the LYSA (The Lymphoma Study Association).

All patients gave written non-opposition consent. Patients samples were allocated to GAPDH high and low groups according to their respective expression (see GAPDH IHC staining and scoring section).

Training cohort. A total of 43 patients aged >18 years (aged between 24-85 years) who had been diagnosed with de novo DLBCL and then treated with Rituximab and CHOP between 2008 and 2014 at Onco-Hematology Department of Nice CHU (France) and Saint-Louis Hospital, APHP, Paris (France) were included in this training set. The samples were prepared from the paraffin blocks of lymph nodes that were recovered from the archives of the Pathology departments.

Validation cohort. All cases analyzed in the validation cohort were enrolled in the prospectively randomized phase 3 multi-center GELA-LNH03-6B trial [START_REF] Delarue | Dose-dense rituximab-CHOP compared with standard rituximab-CHOP in elderly patients with diffuse large B-cell lymphoma (the LNH03-6B study): a randomised phase 3 trial[END_REF] in which 602 patients with untreated diffuse large B-cell lymphomas (DLBCLs) and at least one adverse prognostic factor aged between 60-80 years had been randomly assigned to receive eight cycles of R-CHOP-14 or eight cycles of R-CHOP-21. They did not show any difference in efficacy endpoints between dose-dense and standard regimens of R-CHOP. 294 biopsies specimens at diagnosis were available for the tissue microarray (TMA). TMA contains three representative 0.6-mm cores of routinely processed tissues

GAPDH immunohistochemical (IHC) staining and scoring.

Sections (4 µm) of formalin-fixed, paraffin-embedded DLBCL biopsies (training cohort) or tissuemicroarray (TMA) from the LYSA group (validation cohort) were treated for deparaffinization, rehydration, and antigen retrieval using standard procedures (EnVision™ FLEX reagents, Agilent, Dako). Before immunohistochemistry, antigen retrieval was performed at pH 9.0, for 20 minutes at 97°C, according to the manufacturer's instructions (EnVision™ FLEX reagents, Agilent, Dako). After washing in diluted EnVision FLEX Wash Buffer, slides were incubated in EnVision FLEX Peroxidase-Blocking Reagent solution for 5 min to quench endogenous peroxidase. Slides were then washed, stained at room temperature on an automated system (Autostainer link 48, Dako) with diluted (EnVision FLEX antibody diluent) anti-GAPDH (rabbit polyclonal anti-human GAPDH, HPA40067, Sigma-Aldrich; dilution 1:500) for 20 minutes, washed, incubated 20 minutes with Dako EnVision™ FLEX /HRP), washed, incubated 8 minutes with EnVision FLEX Substrate Working solution and counter-stained with Hematowylin (EnVision™ FLEX Hematoxylin). Sections were then dehydrated and mounted with pertex (Histolab).

Cytoplasmic GAPDH IHC staining was blindly scored by two pathologists from the LCAP from Nice CHU (Dr D. Ambrosetti and Dr. J. Reverso-Meinietti) based on two parameters: the GAPDH IHC staining intensity in tumor cells (scored 0 for absence of staining, 1 for a weak staining, 2 for a medium staining and 3 for a strong staining) and the proportion of tumor cells displaying each intensity. Then GAPDH IHC score was obtained by multiplying each GAPDH staining intensity with the corresponding percentage of tumor cells stained (example: GAPDH IHC score= 0 (non-stained) x % tumor cells + 1 (weak intensity) x % tumor cells stained + 2 (medium intensity) x % tumor cells stained + 3 (strong intensity) x % tumor cells stained). For the training cohort, GAPDH IHC staining was scored on the whole slide. For the TMA, GAPDH IHC score represents the mean of three different tissue spots from the same tumor. GAPDH IHC cut off of 150 was determined using positional scanning [START_REF] Lipton | Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization-positive patients with metastatic breast cancer treated with trastuzumab[END_REF]. Illustrations of GAPDH IHC staining were performed using SlidePath Digital Image software (Leica Biosystems).

Immunohistochemistry and fluorescence in situ hybridization

Sections (4 µm) of formalin-fixed, paraffin-embedded DLBCL biopsies (training cohort) were treated for deparaffinization, rehydration, and antigen retrieval using standard procedures (EnVision™ FLEX reagents, Agilent, Dako). Antibodies against CD5 (M3641, Agilent, Dako), CD79A (M7050, Agilent, Dako), CD20 (M0755, Agilent, Dako), CD10 (GA64861-2, Agilent, Dako), BCL2 (M0887, Agilent, Dako), BCL6 (M7211, Agilent, Dako), MUM1 (M7259, Agilent, Dako), c-MYC (ab32072, Abcam) and phospho-p70S6K (T389) (rabbit monoclonal anti-phospho-p70S6K (T389), #9234, Cell Signaling Technology) were used for immunostainings performed on an automated system (Autostainer link 48, Dako). Antigen retrieval was performed at specific pH prior incubation with indicated antibodies: pH 6.0 prior anti-CD79A, anti-CD20, anti-MUM1 and pH 9.0 prior anti-CD5, anti-CD10, anti-BCL2, anti-BCL6, anti-c-Myc and anti-phospho-p70S6K (T389). Primary antibodies were diluted in EnVision FLEX antibody diluent (anti-CD5, anti-CD10, anti-CD20, anti-BCL2 are prediluted; anti-BCL6, dilution 1:50; anti-phospho-p70S6K (T389) 1:50; anti-CD79A, dilution 1:100; anti-MUM1, dilution 1:100; anti-c-Myc, dilution 1:100) (see IHC procedure in "GAPDH immunohistochemical (IHC) staining and scoring" section). Tissue-microarrays (TMA) from the LYSA group (validation cohort) were treated and immunoassayed for the same proteins using standard procedures of the LYSA platform as previously described [START_REF] Molina | Young patients with non-germinal center B-cell-like diffuse large B-cell lymphoma benefit from intensified chemotherapy with ACVBP plus rituximab compared with CHOP plus rituximab: analysis of data from the Groupe d'Etudes des Lymphomes de l'Adulte/lymphoma study association phase III trial LNH 03-2B[END_REF]. GCB vs non-GCB scoring according to the Hans algorithm [START_REF] Hans | Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray[END_REF], as well as MYC and BCL2 were evaluated [START_REF] Petrella | BCL2 expression but not MYC and BCL2 coexpression predicts survival in elderly patients with diffuse large B-cell lymphoma independently of cell of origin in the phase 3 LNH03-6B trial[END_REF]. In 30 out of the 294 cases, COO classification by IHC (Hans algorithm) could not be assessed. The thresholds employed were 40% for MYC and 70% for BCL2 as previously reported [START_REF] Green | Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone[END_REF][START_REF] Johnson | Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone[END_REF].

FISH analyses were performed on 3µm tissue sections using split signal FISH DNA probes for cMYC/8q24 (Y5410, Dako), BCL2/18q21 (Y5407, Dako), BCL6/3q27 (Y5408, Dako) and slides were analyzed as previously described [START_REF] Copie-Bergman | Immuno-fluorescence in situ hybridization index predicts survival in patients with diffuse large B-cell lymphoma treated with R-CHOP: a GELA study[END_REF]. Scoring of the hybridization signals were performed according to the algorithm published by [START_REF] Haralambieva | Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections[END_REF].

ATP analysis

ATP was measured using the Cell Titer Glo kit (G7570, Promega). Briefly, 20.000 cells were resuspended in 80 µL of corresponding medium supplemented with 10% FBS and distributed in a 96 well plate. Cells were then treated in triplicates for one hour with PBS (control), or sodium iodoacetate (100 µM) to inhibit glycolysis, or a combination of 100 µM of iodoacetate (I9148, Sigma-Aldrich) and 10 µg/ml of oligomycin (O4876, Sigma-Aldrich) to obtain the residual amount of ATP. Short time treatment (1h) was chosen to avoid a metabolic switch upon inhibition of glycolysis and to avoid cell death upon inhibition of both metabolic pathways. 100 µL of Cell Titer Glo reaction mix was then added to each well for a final volume of 200 µL. Plates were analyzed for luminescence with a Luminoscan (Berthold Technologies). We verified that ATP measurements were in the linear range of the detection. The difference between total ATP production and the ATP produced under iodoacetate treatment results in glycolytic ATP contribution. Glycolytic ATP is represented by percentage of total ATP produced by the cells.

Lactate measurement

Lactate concentration in the supernatant was determined by an enzyme-based assay using 900 µM β-NAD + (N1636, Sigma-Aldrich), 175 µg/ml L-lactic dehydrogenase (L2500, Sigma-Aldrich) and 100 μg/ml glutamate-pyruvate transaminase (#10737127001, Roche) diluted in a 620 mM sodium carbonate (S2127, Sigma-Aldrich)-79 mM L-glutamate (G1626, Sigma-Aldrich) buffer adjusted to pH 10. Lithium lactate (L2250, Sigma-Aldrich) was used as a standard. Measurements were recorded from a microplate reader (excitation 355 nm, emission 612 nm) after 30 min incubation at 37 °C.

Lactate secretion was expressed as mM and normalized to viable cell numbers (using Trypan blue exclusion method).

Lactate concentration in the supernatant was also determined electro-enzymatically using the YSI 2950 Biochemistry Analyzer (Yellow Springs Instruments) and normalized to viable cell number.

Glutamine consumption

Cells were grown in DMEM high glucose, no glutamine (#11960044, Thermo Fisher Scientific) supplemented with 2 mM of L-Glutamine (#25030081, Thermo Fisher Scientific), 10% FBS, 50 µM of 2-mercaptoethanol (#31350010, Thermo Fisher Scientific), 0.37 mM of L-asparagine (A0884, Sigma-Aldrich) and 10 mM of HEPES pH 7.4 (#15630056, Thermo Fisher Scientific) for 24 hours.

The concentrations of L-Glutamine (mg/L) from cultured-cell supernatants were determined electroenzymatically using the YSI 2950 Biochemistry Analyzer (Yellow Springs Instruments) and normalized to viable cell number.

L-[ 14 C]-Glutamine uptake

Cells (5x10 6 , in triplicate for each lymphoma) were washed with PBS and incubated 5 min at 37°C in 80 µl of pre-warmed DMEM low glucose (5.5 mM), amino acid-, pyruvic acid-HCO3 --free (US Biological, D9800-13) supplemented to a final concentration of 15 mM D-glucose (G6152, Sigma-Aldrich), 20 mM HEPES (#15630056, Thermo Fisher Scientific) and adjusted to pH 7.4, prior addition of 1.25 µCi/ml of L-[ 14 C]-Glutamine (NEC451050UC, PerkinElmer,) for 30 min at 37°C in a CO2-free incubator. Subsequently, cells were washed twice with PBS and lysed with 120 µl of 0.1N NaOH and mixed with 3 ml of Ultima Gold (#6013321, PerkinElmer). Radioactivity was measured using a β-scintillation counter. Inhibition experiments were performed in the presence of 10 mM of the system A transporter inhibitor α-(Methylamino)isobutyric acid (MeAIB, M2383, Sigma-Aldrich).

2-NBDG uptake

200.000 cells were incubated in glucose-free DMEM medium (#11966025, Thermo Fisher Scientific) containing 40 µM 2-NBDG (#23002-v, Peptide Institute) for 2 hours and analyzed by flow cytometry (Miltenyi Biotec) in the presence of 0.5 µg/ml of DAPI (D9542, Sigma-Aldrich) to represent glucose uptake (Mean Fluorescence intensity) in viable cells.

Enzymatic spectrophotometric measurements of the OxPhos respiratory chain complex I, complex II and citrate synthase were performed at 37°C on cells according to standard procedures [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF]. Proteins were measured according to Bradford microassay (#500-006, Bio-Rad). Results are expressed as activity ratios compared to the activity of citrate synthase.

Cell viability assays

Cells (0.3×10 6 ) were seeded in 96 well-plates in the presence or absence (Ctl, DMSO or NaCl 0.9%) of phenformin (P7045, Sigma-Aldrich), E-Coli L-asparaginase (Kidrolase®, Jazz Pharmaceutical), Rapamycin (#1292, Bio-techne) or anti-CD20 antibody Rituximab (Mabthera®, #9197719, Roche) at indicated concentration and period of time. Cells were then labeled with 0.5 µg/ml of DAPI (D9542, Sigma-Aldrich) and analyzed immediately by flow cytometry (Miltenyi Biotec).

Activation of the B-cell receptor

SU-DHL-6 cells (1.10 6 cells/ml) were incubated in the presence of 10 µg/ml of anti-IgM antibody (#109-006-129, Jackson ImmunoResearch) for 10 min and 18hours.

Isolation of human tumor cells from DLBCL core-needle biopsies

'Fresh' image-guided core-needle biopsies (non-frozen tumor biopsies) were harvested in the radiology departments of St Louis hospital in Paris (APHP, France), Centre Hospitalier Princesse Grace (CHPG, Monaco) and the Centre Antoine Lacassagne (CAL, Nice, France), anonymized and immediately sent to our laboratory (Nice, France) in accordance with the GlyPhome protocol approved by institutional review boards of APHP, CHPG and CAL and Commission Nationale de l'Informatique et des Libertés (CNIL) reference 912657 V1. All patients gave written non-opposition consent. Immediately upon receipt, tumor cells were dissociated in RPMI medium (#61870044, Thermo Fisher Scientific) supplemented with 2% of FBS, 10 µg/ml of DNAse (D4627, Sigma-Aldrich) and 0.1 mg/ml of collagenase (C9891, Sigma-Aldrich) 5 min at 37°C. Red blood cells were lyzed (BD Pharm Lyse, #555899, BD Bioscience) 1 min at room temperature. After PBS washing, cells were counted (by flow cytometry, using DAPI staining) and analyzed for glycolytic ATP production in RPMI medium (#61870044, Thermo Fisher Scientific) supplemented with 10% FBS.

Diagnosis of the biopsies was provided by histopathology analyses.

Plasmids and generation of GAPDH-overexpressing cells and gapdh-silenced cells.

The coding sequence of human gapdh (M33197) was first cloned by PCR into a pcDNA3.1/V5-His TOPO TA expression plasmid (K480001, Invitrogen) following classical methods. gapdh was then subcloned into a pMIG vector (9044, Addgene) for retroviral infection.

The shRNA targeting mouse gapdh was obtained using the oligonucleotide sequences forward 5'-GATCCCCGTATGATGACATCAAGAAGTTCAAGAGACTTCTTGATGTCATCATACTTTTTA-3' and reverse 5'-AGCTTAAAAAGTATGATGACATCAAGAAGTCTCTTGAACTTCTTGATGTCATCATACGGG-3' and inserted into the pSUPER-retro.neo+ gfp vector (VEC-PRT-0006, Oligoengine) following the manufacturer's instruction. Self-inactivating retroviruses were generated by transient transfection of 293T cells (ATCC, #CRL-1573) and tittered as described previously [START_REF] Frecha | In vivo gene delivery into hCD34+ cells in a humanized mouse model[END_REF]. Briefly 8.6 µg of plasmid of interest was co-transfected with 3 µg of envelope plasmid phCMV-VSV-G, 8.6 µg of MLV-Gag-Pol (gift from Dr. Verhoeyen) using the classical calcium phosphate method. 72 hours after transduction, GFP positive cells were sorted (FACS Aria, BD Biosciences).

RNA extraction and Real-time Quantitative PCR

Total RNA was extracted from cells (RNeasy mini kit, #74104, Qiagen) according to the manufacturer's instructions. 2µg of RNA were added to 20 µl reverse transcription-PCR using the Omniscript RT kit (#205113, Qiagen). The relative mRNA levels of gapdh expression (mouse or human) were obtained by real-time quantitative PCR (qPCR), using the TaqMan Fast Universal PCR Master Mix (#4366072, Fisher Scientific) and TaqMan assay primer sets (mouse gapdh:

Mm99999915_g1; human gapdh: Hs02758991_g1, Thermo Fisher Scientific) on the Step One (Applied Biosystems) according to the manufacturer's instructions. For mouse primary malignant B cells, samples were normalized to mouse rn18s (Mm03928990_g1, Thermo Fisher Scientific). For human DLBCL cell lines, samples were normalized to human rplp0 (Hs99999902_m1, Thermo Fisher Scientific).

To measure gapdh mRNA stability, cells were treated with 5 µg/ml of Actinomycin D (A9415, Sigma-Aldrich) for indicated period of times.

Western blot analysis

Cells were washed in PBS and lysed in laemmli buffer. Tissue samples were collected and lysed in Precellys 24 (Bertin Instruments) homogenizer (3x30 s, 6500g) in laemmli buffer. After quantification sheath gas flow was set to 20 units, the auxiliary gas flow was set to 5 units, and the sweep gas flow was set to 0 unit. The metabolites were detected across a mass range of 75-1,000 m/z at a resolution of 35,000 (at 200 m/z) with the AGC target at 106, and the maximum injection time at 250 ms. Lock masses were used to insure mass accuracy below 5 ppm. Data were acquired with Thermo Xcalibur software (Thermo Fisher Scientific). The peak area of metabolites was determined using Thermo

TraceFinder software (Thermo Fisher Scientific), identified by the exact mass of each singly charged ion and by known retention time on the HPLC column. Statistical analyses were performed using Metaboanalyst 4.0 software.

H HRMAS NMR spectroscopy

Cell pellets (10 million cells) were frozen at -80°C. After thawing cell pellets, 10 microliters of D2O were added. Solvent and cells were then placed into a 30 µl disposable insert introduced into a 4mm

ZrO2 HRMAS rotor before NMR analysis. All the NMR experiments were recorded on a Bruker

Avance III spectrometer operating at 400 MHZ and 100MHZ for 1 H and 13 C, respectively, and equipped with a 1 H/ 13 C/ 31 P HRMAS probe. All the spectra were recorded at 4000 Hz spinning rate and a temperature of 277 K. For each sample, 1 H HRMAS NMR spectrum was acquired using the Carr-Purcell-Meiboom-Gill NMR sequence, preceded by a water presaturation pulse during relaxation time of 2 s. This sequence enables us to reduce the macromolecule and lipid signal intensities using a T2 filter of 50 ms synchronised with the rotor rotation speed. For each spectrum, 256 free induction decays of 20 800 data points, were collected using 8000 Hz of spectral window. The FIDs were multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz and zero-filled prior to Fourier transformation. The calibration on the alanine doublet (δ=1.47 ppm) and the phase and baseline corrections were performed manually for all the spectra. The identification of the NMR signals was carried out using 1 H-1 H TOCSY, 1 H-13 C HSQC NMR experiments, online databases (HMDB, BRMB) and literature [START_REF] Nicholson | 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma[END_REF][START_REF] Wishart | Abbreviations: DE, double expressors (Myc-Bcl2); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; R-CHOP, Rituximab with cyclophosphamide, doxorubicin, oncovin and prednisone. n.a, not applicable. Number of samples that could not be analyzed[END_REF]. Each spectrum was divided into 0.005 ppm-width integrated buckets and normalized to total spectrum intensity after having removed the presaturated water signal region (4.85 -5.10 ppm) using AMIX software (Bruker Biospin GmbH, Karlsruhe, Germany). The water region must be removed due to inconsistent signal suppression.

A matrix of 42 samples and 1700 buckets was then created and exported to the software Simca-P v13 (Umetrics, Umea, Sweden) for statistical analysis. Principal component analysis was first performed in order to detect intrinsic clusters and outliers within the dataset. Since expected discrimination was not achieved using PCA, the data were analyzed with orthogonalized projections to latent structures discriminant analysis (OPLS-DA). The OPLS algorithm derives from basic partial least-squares (PLS)

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Pvalue<0.00001 N=125 N=108
13.5

  are sensitive to the inhibition of mitochondrial ETC complex I. Identification of a novel biomarker predicting DLBCL sensitivity to current treatment is only of clinical interest if we can propose new therapeutic options. Since R-CHOP low responders, i.e. DLBCL-GAPDH low , primarily rely on OxPhos metabolism (Fig 1H, 1I), we investigated their sensitivity to inhibitors of mitochondrial energetics.

  Fig S3D). Cells from Eµ-Myc-GAPDH low and Eµ-Myc-GAPDH high lymphomas produce equivalent amount of ATP (Fig 2A). However, Eµ-Myc-GAPDH low cells produce less glycolytic ATP (Fig 2B), as observed in human DLBCL cells (Fig 1H and 1I) and secrete less lactate than Eµ-Myc-GAPDH high cells (Fig 2C). Accordingly, Eµ-Myc-GAPDH low cells consume more O2 (Fig 2D), while glucose transporters expression (Fig S3E) and the mitochondrial mass (Fig S3F) are similar between the two groups.

  , rapamycin does not induce cytotoxic responses (Fig 3H), but rather cytostatic effects, as we observed (Fig S5H). Finally, in vivo inhibition of mTORC1 activity with Temsirolimus (Dancey, 2010) significantly increases the survival of OxPhos Eµ-Myc-GAPDH low -bearing mice (Figs 3I and 3J).

  ). Extracellular sources of Gln are required for Eµ-Myc-GAPDH low cells to sustain basal O2 consumption, maximal respiratory capacities and ATP-coupled OCR (Figs S6E to S6G). Pharmacologic hydrolysis of extracellular sources of Lglutamine and L-asparagine with E-Coli L-asparaginase (Fig 4E), reduces mTORC1 activity (Fig S6H, (Willems et al., 2013)) and induces cytotoxic responses in Eµ-Myc-GAPDH low lymphoma cells (Fig 4F). In addition, gapdh-silenced Eµ-Myc-GAPDH high lymphoma cells are more sensitive than control cells to E-Coli L-ase-induced death (Fig S4E). In vivo, plasma sources of Gln were depleted in E-Coli L-ase treated mice, regardless of the Eµ-Myc lymphoma injected (Fig 4G). However, only the survival of Eµ-Myc-GAPDH low -bearing mice was enhanced upon E-Coli L-ase treatment (Figs 4H and

  GAPDH overexpression was sufficient to reduce mTORC1 signaling (Fig 5A) and to induce a metabolic switch toward glycolysis, as judged by the increase in glycolytic ATP, lactate secretion along with the reduction in baseline OCR and Gln consumption (Figs 5B to 5F). Equivalent observations were obtained upon GAPDH expression in the OxPhos-DLBCL cell line Karpas 422 (Figs S7A to S7C). Importantly, the metabolic switch induced by GAPDH overexpression prevented most of the beneficial effects brought by either phenformin or E-Coli L-asparaginase in vivo (Figs 5G to 5I).

Fig

  Fig 6C illustrates the patient#1, a 24-year-old man who presented a DLBCL in therapeutic failure. At

  Figure titles and legends

C.

  Illustration of IHC staining of GAPDH in germinal center (GC) B cells of a reactive lymph node (LN) (score = 80) and in de novo DLBCL expressing low (score = 80) or high (score = 260) levels of GAPDH protein. Magnification 20X (100 µm). D. Distribution of GAPDH protein expression scored by IHC in GCB cells from human paraffinembedded reactive lymph nodes (n=5) or newly diagnosed DLBCLs expressing low (score ≤ 150; n=15) or high (score > 150; n=28) GAPDH protein (Training cohort). p value from Wilcoxon rank sum. E. OS of R-CHOP-treated DLBCL patients according to the levels of GAPDH expression determined by IHC in newly diagnosed DLBCLs (n=15 GAPDH low and n=28 GAPDH high ; Training cohort). p value from log-rank test. F. Distribution of GAPDH protein expression scored by IHC staining of GAPDH in tissue-microarray corresponding to 294 paraffin-embedded de novo DLBCL biopsies. 204 DLBCLs-GAPDH low and 90 DLBCLs-GAPDH high are identified (validation cohort). p value from Wilcoxon rank sum. G. OS of 294 R-CHOP-treated patients with DLBCLs grouped on the basis of GAPDH expression levels presented in F. p value from log-rank test. H. Transcript abundance (probe intensity) of gapdh mRNA according to the CCC classification (n=82 BCR-DLBCL and n=71 OxPhos-DLBCL) identified by genomic clustering

  , related to Figure1.

Figure 2 .

 2 Figure 2. Mouse primary B lymphomas expressing low levels of GAPDH rely on OxPhos

  From panels A. to E., ***p<0.001 indicated significance of the observed differences. ns, not significant.See also FiguresS3 and S4, related to Figure2.

Figure 3 .

 3 Figure 3. Active mTORC1 signaling in B lymphomas expressing low levels of GAPDH is

Figure 4 .

 4 Figure 4. Mouse primary B lymphomas expressing low levels of GAPDH rely on glutamine

Figure 5 .

 5 Figure 5. Specific overexpression of human GAPDH reduces mTORC1 signaling and decreases

  A. was determined with Clarck electrode, 24 hours after cell seeding. Data are expressed as mean ± s.d (n=3 independent experiments). F. Glutamine consumption was determined 24 hours after cell seeding in the supernatant of Eμ-Myc-GAPDH low cells (lymphoma #F) stably transduced with control (pMIG) or GAPDH-V5-encoding pMIG vectors. Data are expressed as mean ± s.d (n=3 independent experiments). G. Weight of inguinal (left) and axillary (right) lymphomas harvested from wild-type syngeneic C57BL/6 mice twenty days after intravenous injection of primary Eμ-Myc-GAPDH low cells stably over-expressing or not (pMIG) GAPDH-V5. Phenformin (100 mg/kg/day) or vehicle (H2O) were administrated every day for the last thirteen days (pMIG-Vehicle, n=8; pMIG-Phenformin, n=9; GAPDH-V5-Vehicle, n=10; GAPDH-V5-Phenformin, n=7). H. I. Survival of syngeneic C57BL/6 mice intravenously injected with primary Eµ-Myc-GAPDH low cells stably transduced with control pMIG vector (H.) or GAPDH-V5-encoding pMIG vectors (I.) and treated six days later with either vehicle (NaCl 0.9%) or 2500 IU/kg of E-Coli L-asparaginase (L-ase) intraperitoneally five times per week the first week and three times per week the following weeks (n=10 mice/group, p value from log-rank, **p< 0.01; ***p< 0.001). From panel A. to G., *p<0.05, **p<0.01, ***p<0.001 indicated significance of the observed differences. ns, not significant. See also Figure S7, related for Figure 5.

Figure 6 .

 6 Figure 6. Patients with DLBCL-GAPDH low are sensitive to specific inhibitors of mitochondrial

(

  Pirece BCA Protein Assay kit, #23225, Thermo Fisher Scientific), 40 µg of proteins were separated on 8% to 13% SDS polyacrilamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blotted with antibodies against indicated proteins. Immunoreactive bands were detected with an anti-mouse (#7076, Cell Signaling Technology) or an anti-rabbit (7074S, Cell Signaling Technology) IgG horseradish peroxidase (HRP)-linked antibodies. Immunoblots were visualized (FUJIFILM LAS4000) by chemoluminescence using Pierce ECL Western Blotting substrates (#32106, Pierce ECL, Thermo Fisher Scientific). When indicated, Western blot quantification was performed using ImageJ software.Anti-V5 (R96025) was purchased from Invitrogen. Anti-Erk2 (sc-1647) was purchased from Santa Cruz. Anti-GAPDH (ab9485), anti-LDH (ab52488), anti-LDH-B (ab85319), anti-GLS (ab93434) were purchased from Abcam. Anti-HKII (#2106), Anti-ENO1 (#3810), anti-PKM2 (#3198), anti-p53 (#2524), anti-phospho-ACC (Ser 79) (#11818), anti-ACC (3676), anti-phospho-p70-S6K (T389) (#9234), anti-p70S6K (#9202), anti-phospho-Syk (Tyr 525/526) (#2710), anti-Syk (#2712) were purchased from Cell Signaling Technology.

  Figure 1 Chiche J et al.

  Figure 2 Chiche J et al.

Figure 6

 6 Figure 6 Chiche J et al.

Table 1 . GAPDH expression levels are not associated with other biological prognostic factors.

 1

	TRAINING COHORT (n=43)	VALIDATION COHORT (n=294)

Acknowledgements

We gratefully acknowledge the C3M animal facility, the Tumorothèque of Hôpital Saint-Louis and the Lymphoma Study Association (LYSA). We thank the technician of the Laboratoire Central d'AnatomocytoPathologie (LCAP), CHU de Nice, S. Destrée and A. Borderie. We thank I.

Nemazanyy from the Platform for Metabolic Analyses (Neckert-Enfants Malades Institute, Paris). We thank A. Vandenberghe, N. Alet, N. Jourdan-Desrayaud, F. Larbret, F. Reinier, N. Nottet, M. Baia, N. Vailhen, S. Roulland, M. Zala, and I. Ben-Sahra for reagents and input. This work has been supported by the Fondation ARC pour la Recherche sur le Cancer (PGA1 RC-20170205463), the Agence Nationale de la Recherche (LABEX SIGNALIFE ANR-11-LABX-0028-01), the Fondation de France, Centre Scientifique de Monaco, the Cancéropôle PACA and INSERM transfert. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 766214 (Meta-Can). C.T. is funded by the Nella and Amadeus Barletta foundation (FNAB). BN is funded by Cancéropole PACA, la Fondation ARC (PGA120150202381) and Plan cancer 2015 ITMO cancer (C15077AS). The IPMC functional genomics platform is a member of the "France Génomique" consortium (ANR-10-Infra-01), with financial support from the Cancéropôle PACA. E.Vil. is supported by La Ligue contre le Cancer; J.C. is supported by foundation Tourre and France Lymphoma Espoir, G.B is supported by la Fondation pour la Recherche Médicale (FRM, FDM20150734500) and J R-M. is supported by INCa.

Data analysis and Software availability

Code availability

R code for microarray analysis of Lenz G. et al. data set is available at doi:10.17632/f4f3wbszmd.1

Author Contributions

JC performed the majority of research described herein and was assisted by AM, C R-P, R M, E Vil., JPB, EP, MGR, KF, SM, E Ver., V P-F, LS, GB, BN. Clinical study and patient samples: J R-M, JB, DA, J-FM, TJM, CC-B, JL-C, IP, FP, EDK, BT, GG, VD, CD-D, RD, AB, TP, NM, CT.

Bioinformatic analysis: AP, PB. J-E.R secured funding and supervised the study. CT supervised the clinical study. JC and J-E.R designed research and wrote the manuscript. mice were sacrificed two hours after the last gavage of phenformin to analyze effects of the compound on lymphoma cell signaling. E-Coli L-asparaginase (L-ase) treated mice were sacrificed four hours after the last ip injection of L-ase and plasma was immediately isolated to analyze plasma glutamine concentrations. Temsirolimus treated mice were sacrificed 24 hours after the last treatment to analyze in vivo efficacy of this compound within the lymphomas.

All mice were maintained in specific pathogen-free conditions and all experimental procedures were approved by the national Animal Care and Use Committee and the regional ethics committee (05111.02 from Direction Générale de la Recherche et de l'Innovation). Survival was determined as the time between the i.v. injection of the cells and the time when mice had to be sacrificed, as soon as they exhibit systemic signs of illness (apathy, hair loss, breathing problems, precipitous weight loss, and limited ability to reach food or water), in agreement with the guidelines of the Institutional Animal Care and Use Committee. Food was given ad libitum. Experiments using the Eµ-Myc cells transplantation in syngeneic C57BL/6 mice have shown statistical significance starting from 6 mice/group. No randomization was performed. Endpoint was evaluated by blinding.

Cell lines and culture conditions

Mouse primary Eµ-Myc lymphomas cells were isolated from different C57BL/6 Eµ-Myc transgenic mice as previously described [START_REF] Chiche | GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin's B lymphomas via NF-kappaB-dependent induction of HIF-1alpha[END_REF] and maintained in DMEM-GlutamaX (#31966047, Thermo Fisher Scientific) supplemented with 10% FBS, 50 µM of 2-mercaptoethanol (#31350010, Thermo Fisher Scientific), 0.37 mM of L-asparagine (A0884, Sigma-Aldrich) and 10 mM of HEPES pH 7.4 (#15630056, Thermo Fisher Scientific).

The human DLBCL cell lines Toledo, Karpas 422, SU-DHL-4 and SU-DHL-6 were tested mycoplasma-free. Cell line authentication was performed by DSMZ by DNA profiling using 8 different and highly polymorphic short tandem repeat (STR) loci. There was also no presence of mitochondrial DNA sequences from rodent cells as mouse, rat, Chinese and Syrian hamster. The Karpas 422 cell line was from the ECACC (European Collection of Authenticated Cell cultures), the Toledo cell line was from ATCC (American Type Culture Collection), the SU-DHL-4 and SUD-HL-6 cell lines were from DSMZ (German Collection of Microorganisms and Cell Culture). Toledo, Karpas 422 and SU-DHL-4 cells were maintained in RPMI-GlutamaX (#61870044, Thermo Fisher Scientific), supplemented with 10% FBS. SU-DHL-6 cells were maintained in RPMI-GlutamaX supplemented with 20% FBS.

Microarray data analysis

Raw.CEL files and clinical information from the study of [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF]. were downloaded from the Gene Expression Omnibus database (GSE10846). The total number of samples included in this study was 414, 181 treated with CHOP and 233 with R-CHOP. Data were summarized, normalized and log2 transformed using the justRMA function from the Bioconductor package affy (version 1.40.0, [START_REF] Gautier | affy--analysis of Affymetrix GeneChip data at the probe level[END_REF]). Follow-up time was limited to 5 years.

Association between gene expression levels and overall survival was performed on samples from the R-CHOP arm only. Samples with low expression levels were filtered out (log2 intensity < 8). For each remaining probeset, samples were split into two groups (below and above the median) and difference in survival was assessed using Cox proportional hazards models. 587 candidate probesets were selected based on nominal p-value < 0.05 and an abs(log2 HR)>0.75.

Association of expression levels with OS was first tested by categorizing samples into high/low expression samples using median values. Then for gapdh, cutoff was optimized using positional scanning [START_REF] Lipton | Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization-positive patients with metastatic breast cancer treated with trastuzumab[END_REF]. Survival curves were plotted following the Kaplan and Meier method, and statistical significance was assessed using the log-rank test. Survival analysis was performed using functions from the R package survival.

OxPhos and BCR classification were obtained from the supplemental data of [START_REF] Caro | Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma[END_REF].

Statistical significance was assessed using Wilcoxon rank sum test. Enrichment in biological themes (Molecular function and canonical pathways) were performed using Ingenuity Pathway Analysis (Beecher Instruments, Silver Spring, MD).

Clinical protocol KTM (Kidrolase-Torisel-Metformin)

Four patients harboring de novo Myc-translocated-DLBCLs and selected for KTM treatment presented i) a retrospective GAPDH low levels (scored by IHC in diagnosed biopsies), ii) a therapeutic failure after a median of 2 prior lines of Rituximab-based therapies and iii) adequate performance status and organ functions. KTM treatment consisted in L-asparaginase (K, Kidrolase®, Jazz Pharamaceutical, 6000 UI/m 2 ) on days 1, 3, 5, 7, 9, 11, and 13, mTOR inhibitor (T, Torisel, Pfizer, 75 mg) on days 1, 7 and 14, and Metformin (Mylan, 1000 mg/day) on day 14 to day 28. The study was approved on Oct 16 th , 2012 by the Scientific Committee of the LYSA (The Lymphoma Study Association, reference: REMtor). All patients gave a written informed consent to receive the treatment. Consent to publish patients' images was obtained. There was no clinical trial registration number because the therapeutic protocol was conducted on a local cohort.

Methods details

COO classification

Frozen tissues were analyzed using GeneChip Human Genome HGU133 Plus 2.0 arrays (Affymetrix, Santa Clara) as previously reported [START_REF] Dubois | Biological and Clinical Relevance of Associated Genomic Alterations in MYD88 L265P and non-L265P-Mutated Diffuse Large B-Cell Lymphoma: Analysis of 361 Cases[END_REF]. From the LNH03-6B TMA (validation cohort), sixty DLBCLs had both COO molecular classification.

Reverse transcription-multiplex ligation-dependent probe amplification (RT-MLPA) was performed to determine the COO on thirty-five formalin-fixed paraffin-embedded (FFPE) DLBCLs (training cohort), using a targeted molecular gene expression approach as recently described [START_REF] Mareschal | Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study[END_REF].

Measurement of the mitochondrial mass

The mitochondrial mass was measured by flow cytometry (Miltenyi Biotec) in viable (DAPI negative) cells), after 30 min incubation of cells at 37°C in the presence of 150 nM of Mitotracker green probe (M7514, Invitrogen).

Oxygen consumption

Polarographic studies: Intact cell respiration and mitochondrial substrates oxidation on 0.004% digitonin (D5628, Sigma-Aldrich)-permeabilized cells were carried out as previously described [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF]. Proteins were measured according to Bradford microassay (#500-006, Bio-Rad).

Results were expressed in nanomols of oxygen per minute per milligram of proteins or per cell number.

XF Seahorse Analyzer:

Oxygen consumption rate (OCR) was measured in real time using the XF96 extracellular flux analyzer (Agilent). Eµ-Myc cells were seeded on Cell-Tak (#10317081, Thermo Fisher Scientific)-coated XF96 plates at 0.12x10 6 cells/well in 180 µl of XF base medium minimal DMEM media (#102353-100, Agilent) supplemented with 20 mM D-glucose (G6152, Sigma-Aldrich), 1 mM sodium pyruvate (#11360088, Thermo Fisher Scientific), 2 mM L-glutamine (#25030081, Thermo Fisher Scientific) and adjusted to pH 7.4. The plates were spun at 200 g (breaks off) and incubated at 37°C for 20 min to ensure cell attachment. Measurements were obtained under basal conditions and in response to mitochondrial inhibitors, 1 µM oligomycin (O4876, Sigma-Aldrich), 0.5 µM of Carbonyl cyanide 4-(trifluoromethoxy)phenylhudrazone (FCCP, C2920, Sigma-Aldrich), and 1µM rotenone (R8875, Sigma-Aldrich) combined with 2µM antimycin A (A8674, Sigma-Aldrich).

Mitochondrial respiratory complex activities

Flow cytometry analysis

Intracellular staining for GAPDH was performed using the Cytofix/Cytoperm kit (#554714, BD Biosciences), anti-GAPDH (ab9485, Abcam) and Alexa-Fluor 647 goat anti-rabbit IgG secondary antibody (ab150087, Abcam).

IgG and IgM expressions on the surface of DLBCL cell lines were analyzed using FITC-anti-IgG Miltenyi Biotec) and APC-anti-IgM (#130-093-076, Miltenyi Biotec).

Staining were analyzed by flow cytometry (Miltenyi Biotec). Analysis of the FACS data was performed using Macsquantify Version 2.11 (Miltenyi).

Targeted LC-MS metabolites analyses

Metabolites were extracted as described [START_REF] Mackay | Analysis of Cell Metabolism Using LC-MS and Isotope Tracers[END_REF]. Briefly, 1.10 6 Eµ-Myc cells (4 replicates per clones) were used for extraction. Extraction solution was composed of 50% methanol, 30% ACN, and 20% water. The volume of extraction solution added was adjusted to the number of cells (0.5 ml/1x10 6 cells). After addition of extraction solution, samples were vortexed for 5 min at 4°C and then centrifuged at 16,000 g for 15 min at 4°C. The supernatants were collected and stored at -80°C until analyses. Liquid chromatography/Mass Spectrometry (LC/MS) analyses were conducted on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and coupled to a Dionex UltiMate 3000 UPLC system (Thermo, USA). External mass calibration was performed using the standard calibration mixture every 7 days as recommended by the manufacturer. 5 μl of each sample was injected onto Zic-pHilic (150 mm × 2.1 mm i.d. 5 μm) with the guard column (20 mm × 2.1 mm i.d. 5 μm) (Millipore) for the liquid chromatography separation.

Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide (pH 9.2); buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.200 μl/min as follows: 0-20 min; linear gradient from 80% to 20% B; 20-20.5 min; linear gradient from 20% to 80% B; 20.5-28 min: hold at 80% B [START_REF] Mackay | Analysis of Cell Metabolism Using LC-MS and Isotope Tracers[END_REF]. The mass spectrometer was operated in full scan, polarity switching mode with the spray voltage set to 2.5 kV, the heated capillary held at 320°C. The necessary to achieve full relaxation for all the metabolites in order to enable absolute quantification.

For each sample, 128 free induction decays (FID) of 32768 complex data points were collected using a spectral width of 12 000 Hz. A sample of 1.67 mM of maleic acid was used as an external reference to calibrate the ERETIC signal (electronic signal) used for the quantification of glutamine.

Quantification and Statistical Analysis

All survival analyses were performed on an intention-to-treat basis. Categorical variables were compared using the chi-squared or Fisher exact tests. Progression-free survival (PFS) was defined as the time from study entry until disease progression or death. Overall survival (OS) was defined as the time from the start of treatment until death. Survival functions were estimated using the Kaplan-Meier