Fatty acid metabolism provides an essential survival signal in OxPhos and BCR DLBCL cells

doi:10.21203/rs.3.rs-5125394/v1

Download PDF

Article

Fatty acid metabolism provides an essential survival signal in OxPhos and BCR DLBCL cells

https://doi.org/10.21203/rs.3.rs-5125394/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of malignant lymphoma and is a heterogeneous disease with various gene and chromosomal abnormalities. The development of novel therapeutic treatments has improved DLBCL prognosis, but patients with early relapse or refractory disease have a poor outcome (with a mortality of ~ 40%). Metabolic reprogramming is a hallmark of cancer cells. Fatty acid (FA) metabolism is frequently altered in cancer cells and recently emerged as a critical survival path for cancer cell survival. Here, we reveal using a large panel of DLBCL cell lines characterized for their metabolic status that targeting of FA metabolism induces massive DLBCL cell death, regardless of their OxPhos or BCR/glycolytic subtype. Further, FA drives resistance of DLBCL cell death induced by mitochondrial stress upon treatment with either metformin or L-asparaginase, two FDA-approved antimetabolic drugs. Interestingly, combining inhibition of FA metabolism with that of the mTOR oncogenic pathway strongly potentiates DLBCL cell death. Altogether our data highlight the central role played by FA metabolism in DLBCL cell survival, independently of their metabolic subtype, and provide the framework for the use of drugs targeting this metabolic vulnerability to overcome resistance in DLBCL patients.

Biological sciences/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma

Biological sciences/Cell biology/Cell death

Diffuse large B-cell lymphoma (DLBCL) is the most common type of B-cell non-Hodgkin’s lymphoma (NHL), accounting for around 40% of all NHLs worldwide (1, 2). Although standard immunochemotherapy R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) achieves a cure rate of about 60% of DLBCL patients, 40% of DLBCL patients experience relapse, remain refractory to conventional treatment, progress and die (1, 3). Recently, cutting-edge immunotherapies by CAR-T cells have improved outcomes of refractory/relapsed DLBCL patients, but 60% will still succumb (4–6). Therefore, it is necessary to explore novel vulnerabilities and therapeutic strategies for patients with DLBCL. DLBCL is a highly heterogeneous disease with various gene and chromosomal abnormalities, and oncogenic drivers (7–10). DLBCL can be classified into several molecular subtypes identified through coordinated expression of gene clusters (11, 12). The cell of origin (COO) classification has uncovered two molecular subtypes: germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCLs (11). More recently, metabolic signatures uncovered distinct metabolic DLBCL subsets (12), including the OxPhos DLBCL subset that is characterized by a non-functional BCR and an increased mitochondrial metabolism that is required for this DLBCL subset survival, and the BCR subset that presents an up-regulation of genes encoding BCR signaling components and enzymes associated with glycolysis (12–14).

Cancer cells exhibit bioenergetics alterations in order to meet their high-energy requirement, and rewiring of cancer cell metabolism is now recognized as a hallmark of cancer (15). Therefore, targeting tumor energy metabolism has opened up a potential therapeutic window. High glucose consumption through aerobic glycolysis is well known as the “Warburg effect”, but recent discoveries have shown that amino acid as well as lipid and fatty acid (FA) metabolism are also critical for tumorigenesis (16–19). However, how metabolism reprogramming, especially FA metabolism, impacts on DLBCL cell survival and contributes to poor DLBCL patient outcome is still an emerging field.

Fatty acids (FA) are the main building blocks of several lipid species, including phospholipids, sphingolipids and triglycerides and a major source of energy for cancer cell survival via FA β-Oxidation (FAO) (17, 18). Cancer cells are constantly replenished with FA via three main routes that consist in uptake of exogenous FA, de novo FA synthesis and lipolysis from lipid droplets (18, 20–22). How intracellular FA metabolism affects DLBCL cell survival and whether the metabolic status of DLBCL cells impacts on FA-dependent survival remains poorly defined.

In the study presented here, we demonstrated for the first time in a large panel of DLBCL cell lines characterized for their metabolic status that targeting FAO and FA neosynthesis induces massive DLBCL cell death, both in the OxPhos and BCR/glycolytic subtypes. Further, FA drives resistance to DLBCL cell death induced by mitochondrial stress upon treatment with either metformin or L-asparaginase, two FDA-approved antimetabolic drugs. Interestingly, combining inhibition of FA metabolism with the mTOR oncogenic pathway strongly potentiates DLBCL cell death. Altogether our data highlight the central role played by FA metabolism in DLBCL cell survival, independently of their metabolic subtype, and provide the framework for the use of drugs targeting this metabolic vulnerability to overcome resistance in DLBCL patients.

Metabolic characterization of an extended panel of DLBCL cell lines

To explore the contribution of fatty acid metabolism to DLBCL cell survival and its possible relationship with their metabolic and/or BCR status, it was central to use a large panel of DLBCL cell lines whose metabolic status was defined. We used a large panel of eleven different DLBCL cell lines, namely K422, MD901, OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly8, OCI-Ly19, SU-DHL4, SU-DHL5, SU-DHL6 and SU-DHL10. However, since the metabolic status of several of them was still unknown, we first established whether these DLBCL cell lines are OxPhos or glycolytic DLBCL cell lines. We thus evaluated the level of production of glycolytic ATP in all eleven DLBCL cell lines. Four DLBCL cell lines exhibited a low level of glycolytic ATP (MD901, K422, OCI-Ly8 and OCI-Ly19) compared to the seven other DLBCL cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, SU-DHL4, SU-DHL5, SU-DHL6 and SU-DHL10) (Fig. 1A, B). R406 is an inhibitor of the spleen tyrosine kinase (SYK) that only induces apoptosis in DLBCL cell lines having an active BCR signaling, whereas non-BCR DLBCL cell lines do not display functional BCR signaling are insensitive to this inhibitor. Two (SU-DHL5 and OCI-Ly8) out of our eleven DLBCL cell lines have never been evaluated for their R406 sensitivity. As presented in Fig. 1C, OCI-Ly8 was insensitive to R406, whereas SU-DHL5 was highly sensitive to R406 at the same doses. Altogether, our data allowed to classify the large panel of eleven DLBCL cell lines for their metabolic OxPhos vs glycolytic status as well as for their BCR vs non-BCR status (Fig. 1D). Further, since lipid droplets are now well recognized as an important source of fatty acids (FAs) in cancer cells (20, 21), we assessed a potential difference in lipid droplet content in OxPhos vs glycolytic DLBCL cell lines. Interestingly, no significant differences were observed in the lipid droplet content between OxPhos and glycolytic DLBCL cell lines (Fig. 1E, F).

Inhibition of fatty acid oxidation (FAO) induces massive DLBCL cell death, both in ABC and GCB, OxPhos and BCR/glycolytic subtypes

While it is reported that exogenous palmitate increases mitochondrial respiration and survival of OxPhos DLBCL cells (14), whether basal FAO provides (i.e. in the absence of high levels of exogenous FA) an important survival signal in OxPhos and BCR/glycolytic DLBCL cells is currently unknown. We thus evaluated the impact of 4-bromocrotonic acid (4-BrCA) that irreversibly inhibits FAO of long-, medium- and short-chain FAs (23) on OxPhos and glycolytic DLBCL cell lines. Strikingly, 4-BrCA induced a marked dose-dependent induction (from 7.5 to 20 µM) of DLBCL cell death in most tested DLBCL cell lines (Fig. 2A). The two OxPhos/GCB K422 and glycolytic/GCB DLBCL OCI-Ly1 cell lines were particularly sensitive, with almost all DLBCL cells being killed at 7.5 µM after a treatment of only 24 h (Fig. 2A). Interestingly, no significant differences in DLBCL cell survival upon FAO inhibition were observed between OxPhos and glycolytic DLBCL cell lines (Fig. 2B, C). Altogether, these data indicate that blocking FAO induces massive DLBCL cell death independently of current metabolic classification.

Inhibition of FASN induces DLBCL cell death

In contrast to normal cells, de novo lipogenesis is activated in cancer cells, providing selective proliferative and survival advantages (24, 25). Fatty acid synthase (FASN), a key multienzyme complex responsible for de novo lipogenesis, is upregulated in DLBCL patients (26–28). Since the ascription of an oncogenic role for FASN, this enzyme has been considered as a potential therapeutic target for cancer treatment (25, 29). Therefore, we undertook to evaluate how blockage of FASN activity impacts on DLBCL survival. Treatment of the large panel of DLBCL cell lines with C75 led to dose-dependent (from 50 to 100 µM) DLBCL cell death (Fig. 3A). Note that the cell killing induced by C75 is less efficient than 4-BrCA treatment (compare Fig. 2A with Fig. 3A). At the highest dose of C75 (100 µM) there was an average of 60% of cell death, whereas the highest dose of 4-BCA (20 µM) resulted in only 20–30% of DLBCL cell survival after 24h of treatment (Fig. 3A, B). No significant difference of sensitivity to FASN inhibition was observed between OxPhos vs glycolytic DLBCL cells (Fig. 3B, C). Several reversible FASN inhibitors were recently developed by Sagimet Biosciences and have demonstrated antitumor activity in vitro and in vivo, along with some in phase II clinical trials (30). Interestingly, treatment of DLBCL with TVB-3166 strongly impacted on DLBCL survival (Fig. 3D) in a dose-dependent manner, thus reinforcing the beneficial impact of targeting FA neosynthesis. Collectively, our data show that both OxPhos and BCR/glycolytic DLBCL cell survival relies on FASN activity.

Combining inhibition of FA metabolism with mitochondrial stress promotes DLBCL cell death

Metformin is a widely prescribed anti-hyperglycemic biguanide drug for the treatment of type II diabetics, specifically inhibiting complex I of the mitochondrial electron transport chain (31). L-asparaginase (® Kidrolase) catalyzes the hydrolysis of asparagine and glutamine into aspartate and glutamate, respectively, accompanied by mitochondrial dysfunction (32–35). Interestingly, combining L-asparaginase with either the FAO inhibitor 4-BrCA (Fig. 4A) or the FASN inhibitor C75 (Fig. 4B) significantly promoted DLBCL cell death. Similarly, the combination of FA metabolism inhibitors with metformin significantly reduced DLBCL cell survival (Figs. 4A, B). Interestingly, the combination of metformin and L-asparaginase synergizes with lipid metabolism targeting to induce cell death in OxPhos and glycolytic DLBCL cell lines (Figs. 4A, B, M + K).

Collectively, our findings demonstrate that targeting multiple metabolic pathways (lipids, mitochondria, glutamine) impacts more strongly on DLBCL cell survival.

Combination of FASN and mTORC1 inhibition promotes DLBCL cell death

The mTOR oncogenic signaling pathway plays a central role in lipogenesis (36–39) and has been reported to play an important role in DLBCL cell survival (40, 41). Therefore, we have undertaken to evaluate the impact of the mTOR inhibitor rapamycin either alone or in combination with the FA neosynthesis inhibition. mTORC1 inhibition has a strong cytostatic effect (Fig. 5A) but do not induce DLBCL cell death (Fig. 5B, C). Remarkably, combining rapamycin with FASN inhibition promotes DLBCL cell death (Fig. 5B, C), therefore indicating that combining inhibition of mTOR oncogenic pathway and FA metabolism has a broader metabolic impact on DLBCL cells, thereby impacting more strongly of their survival (both OxPhos and glycolytic, ABC and GCB).

Reprogramming of energy metabolism is a well-established hallmark of cancer cells (15). Recently, FA metabolism has emerged as a central player in altered energetics in cancers affecting cancer cell survival (17, 18, 42). FA are described as an energy source through the process of FAO in the mitochondria. The upregulation of FA synthesis is a classically described metabolic alteration in cancer (24, 25, 43). In the study presented here, we conducted the first comprehensive analysis on how inhibition of FA metabolism impacts on DLBCL survival using a large panel of eleven DLBCL cell lines characterized for their metabolic status (OxPhos vs glycolytic) and cell of origin (ABC vs GCB). We uncovered that DLBCL cells are sensitive to drugs targeting either lipid oxidation for energy production, or the lipogenic enzyme FASN, in all subtypes. First, we have shown that FAO inhibition targeting the 3-ketoacyl-CoA thiolase, the enzyme of the last step of β-oxydation (23) induces massive DLBCL cell death in both the OxPhos and glycolytic metabolic subtypes. Second, targeting the lipogenic enzyme FASN using the C75 inhibitor leads to significant DLBCL cell death. Third, the small molecule TVB-3166 developed by Sagimet Biosciences also induces strong DLBCL cell death.

FASN, one of the lipogenic enzymes, has been known for many years to be upregulated in several cancer types, including DLBCL (25, 29). Recently, FASN overexpression has been associated with the development of a more aggressive phenotype and poor DLBCL patient survival (26, 28, 44). Still, the exact molecular mechanisms underlying dysregulation of FA metabolism in DLBCL cells and their relation to the inferior clinical outcome of patients have remained largely elusive, which is worth further investigation. Few previous studies using a very low number of DLBCL cell lines suggested that ABC DLBCL subset may be more sensitive to FASN inhibition, although with seemingly contradictory results (27, 28, 44, 45). Being the first to use a large panel of GCB DLBCL cell lines, our study clearly demonstrates that DLBCL cells of the GCB subtype are sensitive to FASN inhibition.

Our study shows that DLBCL cells are highly sensitive to inhibition of FAO and de novo FA synthesis. Nonetheless the associated molecular mechanisms remain unclear. Interestingly, we have observed a cooperative effect of targeting FASN and mTORC1 activity to induce DLBCL cell death. mTORC1 oncogenic pathway promotes lipogenesis (36, 38). A link between FASN and aberrant protein translation involving mTORC1 signaling has been reported in DLBCL cells (27, 46). In addition, it has been reported that TVB-3166 stimulated cancer cell apoptosis by modulating the mTOR pathway along with altering the lipid profiles (30). Whether the cooperativity between FASN and mTORC1 is linked to altered control of protein translation or other molecular mechanisms in DLBCL remains an open question.

Cancer cells are very adaptable and exhibit an amazing metabolic plasticity in the most adverse conditions (42). Many clinical trials using individual metabolic inhibitors have failed to improve the outcome of DLBCL patients (47–49). This may illustrate the ability of cancer cells to find a metabolic path to reprogram and bypass the targeted metabolic pathway. In this study, we demonstrated that combining inhibition of FA metabolism and induction of mitochondrial stress (metformin and/or L-asparaginase) triggers massive DLBCL cell death. The beneficial combinatory effect was seen in OxPhos and BCR/glycolytic DLBCL cell lines, thus highlighting the benefit of inducing a wide spectrum of metabolic disturbances to promote optimal DLBCL cell death.

In summary, using for the first time a large panel of DLBCL cell lines, we established that drugs targeting FA metabolism induce massive DLBCL cell death, independently of their metabolic status. Further, we show that simultaneous targeting of FA metabolism and other metabolic-linked survival pathways, including glutamine and mitochondrial metabolism, improves DLBCL cell killing. Our data are of great functional importance because they constitute a significant advance in the understanding of metabolic vulnerabilities of DLBCL cells and provide a strong rationale for the use of compounds targeting FA metabolism as adjuvant therapy to enhance current treatment protocols in R/R DLBCL patients. Numerous drugs and small-molecules inhibitors with the goal to inhibit cancer cell lipid metabolism are under development. As this area of research is moving at a rapid pace, there is hope for lipid-targeted therapeutic strategies to find their path in the clinic.

Human DLBCL cell lines and culture conditions

ABC DLBCL cell lines (MD901, OCI-Ly3) and GCB DLBCL cell lines (Karpas 422 (K422), OCI-Ly1, OCI-Ly19, OCI-Ly7, OCI-Ly8, SU-DHL4, SU-DHL5, SU-DHL6, SU-DHL10) were obtained from José Angel Martinez-Climent (Centro de Investigación Médica Aplicada, Pamplona, Spain). All DLBCL cell lines were cultured in RPMI-1640/GlutaMAX medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco), in an incubator at 37°C with 5% CO2.

Reagents

Metformin (PHR1084), 2-deoxy-D-glucose (2-DG, D8375), rapamycin (553211), C75 (C5490), TVB-3166 (SML1694) were purchased from Merck (Darmstadt, Germany), 4-BrCA (A698521) from AmBeed and L-asparaginase (® Kidrolase) was a kind gift from Isabelle Madelaine-Chambrin and Nathalie Jourdan (Service de Pharmacie Centrale, Hôpital Saint-Louis).

Measurement of intracellular ATP content

Intracellular ATP content was measured using an ATP luminescence detection kit (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, 4 x 10⁵ cells were seeded in 100 µL of complete medium. Cells were treated for 1h with or without 20 mM 2-deoxy-D-glucose (2-DG) at 37°C followed by 30 min at room temperature to inhibit glycolysis. At the end of the incubation time, 100 µL of CellTiter-Glo solution was added, and cells were incubated for 10 min at room temperature. The luminescence signal (relative luminescence units (RLU)) was recorded with a microplate reader (Centro LB 960 microplate luminometer, Berthold Technologies, Bad Wildbach, Germany). The difference between total ATP content and ATP produced under 2-DG treatment results in glycolytic ATP.

Apoptosis Assays

DLBCL cells were harvested and washed twice with cold PBS. Cells were resuspended in 1X binding buffer containing Annexin V/PE (BD Biosciences Pharmingen, San Diego, CA, USA) and 4’,6-diaminidino-2-phenylindole (DAPI, Molecular Probes, Life Technologies, Carlsbad, CA, USA) for 15 min at room temperature. The samples were subjected to cytometric analysis with a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and the data were analyzed using the Flowjo v10.2 software (Ashland, OR, USA).

Cell viability assay

The same number of cells (1.0×10⁵ cells/mL) was cultured in 12-well plates for the indicated period of times (24 h and 48 h). Cell viability was then assessed using the trypan blue dye exclusion with stained (non-viable) and unstained (viable) cells.

Lipid droplet content

Cells were stained with 2µM BODIPY 493/503 (Invitrogen, D3922) for 30 min at 37°C in PBS. Then, cells were stained with 1µg/mL of DAPI for 5 min at room temperature to evaluate lipid droplet content in living cells. The samples were subjected to cytometric analysis with a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and the data were analyzed using the Flowjo v10.2 software (Ashland, OR, USA).

Statistical analysis

Statistical significance was assessed using unpaired Student t-test or one-way ANOVA test (Prism 8.0, GraphPad Software, San Diego, CA, USA). A p-value of 0.05 was considered as statistically significant with the following degrees: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Acknowledgments

The authors thank the Centre d’Imagerie Cellulaire et de Cytométrie, Centre de Recherche des Cordeliers UMRS 1138, Paris for FACS analysis. This work was supported by grants from Fondation Nelia et Amadeo Barletta, Switzerland, Institut National du Cancer, Fondation ARC pour la Recherche sur le Cancer, 12 Rounds contre le Cancer, the integrated cancer research center "SiRIC InsiTu : Insights into cancer: From inflammation to tumor" (grant number INCa-DGOS-INSERM-ITMO Cancer_18008), INSERM, Université Paris Cité and IDEX Université Paris Cité (to V.B.), and doctoral fundings from Fondation Nelia et Amadeo Barletta, Switzerland (A.M.) and Société Française d’Hémathologie (A.M.). J.A.M.-C. is supported by Instituto de Salud Carlos III–FIS–CIBERONC (CB16/12/00489) and Arnal Planelles Foundation. GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR-22-CE14-0066 VIVORUSH, ANR-23-CE44-0030 COPPERMAC, ANR-23-R4HC-0006 Ener-LIGHT); Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Joint Programme on Rare Diseases (EJPRD) Wilsonmed; European Research Council Advanced Investigator Award (ERC-2021-ADG, Grant No. 101052444; project acronym: ICD-Cancer, project title: Immunogenic cell death (ICD) in the cancer-immune dialogue); The ERA4 Health Cardinoff Grant Ener-LIGHT; European Union Horizon 2020 research and innovation programmes Oncobiome (grant agreement number: 825410, Project Acronym: ONCOBIOME, Project title: Gut OncoMicrobiome Signatures [GOMS] associated with cancer incidence, prognosis and prediction of treatment response, Prevalung (grant agreement number 101095604, Project Acronym: PREVALUNG EU, project title: Biomarkers affecting the transition from cardiovascular disease to lung cancer: towards stratified interception), Neutrocure (grant agreement number 861878 : Project Acronym: Neutrocure ; project title: Development of “smart” amplifiers of reactive oxygen species specific to aberrant polymorphonuclear neutrophils for treatment of inflammatory and autoimmune diseases, cancer and myeloablation); National support managed by the Agence Nationale de la Recherche under the France 2030 programme (reference number 21-ESRE-0028, ESR/Equipex+ Onco-Pheno-Screen); Hevolution Network on Senescence in Aging (reference HF-E Einstein Network); Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology ANR-18-IDEX-0001; a Cancer Research ASPIRE Award from the Mark Foundation; PAIR-Obésité INCa_1873, the RHUs Immunolife and LUCA-pi (ANR-21-RHUS-0017 and ANR-23-RHUS-0010, both dedicated to France Relance 2030); Seerave Foundation; SIRIC Cancer Research and Personalized Medicine (CARPEM, SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006 supported by Institut National du Cancer, Ministère des Solidarités et de la Santé and INSERM). This study contributes to the IdEx Université de Paris Cité ANR-18-IDEX-0001. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union, the European Research Council or any other granting authority. Neither the European Union nor any other granting authority can be held responsible for them.

Authorship

Contribution: V.B.: conceptualization, methodology, supervision, project administration; A.M., K.K., K.K.-E.: investigation; V.B., A.M., K.K.-E.: formal analysis; J.A.M.-C., I.M., G.K.: reagents, materials, and analysis tools; V.B., A.M.: writing; A.M.: visualization; C.T., critical reading of the manuscript.

Conflicts of interest

J.A.M.-C. declares research funding (unrelated to the present work) from Roche/Genentech, Jannsen, AstraZeneca, Regeneron, Palleon, Priothera and K36-Therapeutics; and holds stock options, patents rights, and royalties in MIMO Biosciences. IM is a consultant for Osasuna Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Hevolution, Institut Servier, Longevity Vision Funds and Rejuveron Life Sciences. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK’s wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9 m, Tusk and Roche, was on the on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK’s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results. The other authors declare no conflict of interest.

Sehn LH, Salles G. Diffuse Large B-Cell Lymphoma. N Engl J Med. 2021;384(9):842–58.
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 2016;127(20):2375–90.
Coiffier B, Lepage E, Brière J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP Chemotherapy plus Rituximab Compared with CHOP Alone in Elderly Patients with Diffuse Large-B-Cell Lymphoma. N Engl J Med. 2002;346(4):235–42.
Westin JR, Kersten MJ, Salles G, Abramson JS, Schuster SJ, Locke FL, et al. Efficacy and safety of CD19-directed CAR‐T cell therapies in patients with relapsed/refractory aggressive B‐cell lymphomas: Observations from the JULIET, ZUMA‐1, and TRANSCEND trials. Am J Hematol. 2021;96(10):1295–312.
Boardman AP, Salles G. CAR T-cell therapy in large B cell lymphoma. Hematological Oncology. 2023;41(S1):112–8.
Brudno JN, Kochenderfer JN. Recent advances in CAR T-cell toxicity: Mechanisms, manifestations and management. Blood Rev. 2019;34:45–55.
Chapuy B, Stewart C, Dunford AJ, Kim J, Kamburov A, Redd RA, et al. Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat Med. 2018;24(5):679–90.
Schmitz R, Wright GW, Huang DW, Johnson CA, Phelan JD, Wang JQ, et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N Engl J Med. 2018;378(15):1396–407.
Wright GW, Huang DW, Phelan JD, Coulibaly ZA, Roulland S, Young RM, et al. A Probabilistic Classification Tool for Genetic Subtypes of Diffuse Large B Cell Lymphoma with Therapeutic Implications. Cancer Cell. 2020;37(4):551–568.e14.
Rosenthal A, Younes A. High grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6: Double hit and triple hit lymphomas and double expressing lymphoma. Blood Rev. 2017;31(2):37–42.
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403(6769):503–11.
Monti S, Savage KJ, Kutok JL, Feuerhake F, Kurtin P, Mihm M, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood. 2005;105(5):1851–61.
Chen L, Monti S, Juszczynski P, Daley J, Chen W, Witzig TE, et al. SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma. Blood. 2008;111(4):2230–7.
Caro P, Kishan AU, Norberg E, Stanley IA, Chapuy B, Ficarro SB, et al. Metabolic Signatures Uncover Distinct Targets in Molecular Subsets of Diffuse Large B Cell Lymphoma. Cancer Cell. 2012;22(4):547–60.
Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646–74.
Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science. 2009;324(5930):1029–33.
Snaebjornsson MT, Janaki-Raman S, Schulze A. Greasing the Wheels of the Cancer Machine: The Role of Lipid Metabolism in Cancer. Cell Metabolism. 2020;31(1):62–76.
Broadfield LA, Pane AA, Talebi A, Swinnen JV, Fendt SM. Lipid metabolism in cancer: New perspectives and emerging mechanisms. Developmental Cell. 2021;56(10):1363–93.
Pavlova NN, Zhu J, Thompson CB. The hallmarks of cancer metabolism: Still emerging. Cell Metabolism. 2022;34(3):355–77.
Petan T, Jarc E, Jusović M. Lipid Droplets in Cancer: Guardians of Fat in a Stressful World. Molecules [Internet]. 2018 Aug 3 [cited 2021 Mar 21];23(8). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222695/
Cruz ALS, Barreto E de A, Fazolini NPB, Viola JPB, Bozza PT. Lipid droplets: platforms with multiple functions in cancer hallmarks. Cell Death Dis. 2020;11(2):1–16.
Zadoorian A, Du X, Yang H. Lipid droplet biogenesis and functions in health and disease. Nat Rev Endocrinol. 2023;19(8):443–59.
El-Aleem SA, Schulz H. Evaluation of inhibitors of fatty acid oxidation in rat myocytes. Biochemical Pharmacology. 1987;36(24):4307–12.
Röhrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer. 2016;16(11):732–49.
Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer. 2007;7(10):763–77.
Danilova OV, Dumont LJ, Levy NB, Lansigan F, Kinlaw WB, Danilov AV, et al. FASN and CD36 predict survival in rituximab-treated diffuse large B-cell lymphoma. J Hematopathol. 2013;6(1):11–8.
Kapadia B, Nanaji NM, Bhalla K, Bhandary B, Lapidus R, Beheshti A, et al. Fatty Acid Synthase induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL. Nat Commun. 2018;9:829.
Zhong X, Liu Z, Luo Q, Li J, Zhang W, Shuang Y. Upregulation of fatty acid synthase in MYC and BCL-2 double-expressor lymphoma. Oncol Lett. 2021;21(4):245.
Migita T, Ruiz S, Fornari A, Fiorentino M, Priolo C, Zadra G, et al. Fatty Acid Synthase: A Metabolic Enzyme and Candidate Oncogene in Prostate Cancer. J Natl Cancer Inst. 2009;101(7):519–32.
Ventura R, Mordec K, Waszczuk J, Wang Z, Lai J, Fridlib M, et al. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression. eBioMedicine. 2015;2(8):808–24.
Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, et al. Efficacy and Metabolic Effects of Metformin and Troglitazone in Type II Diabetes Mellitus. New England Journal of Medicine. 1998;338(13):867–73.
Nuan-Aliman S, Bordereaux D, Thieblemont C, Baud V. The Alternative RelB NF-kB Subunit Exerts a Critical Survival Function upon Metabolic Stress in Diffuse Large B-Cell Lymphoma-Derived Cells. Biomedicines. 2022;10(2):348.
Parmentier JH, Maggi M, Tarasco E, Scotti C, Avramis VI, Mittelman SD. Glutaminase activity determines cytotoxicity of l-asparaginases on most leukemia cell lines. Leukemia Research. 2015;39(7):757–62.
Takahashi H, Inoue J, Sakaguchi K, Takagi M, Mizutani S, Inazawa J. Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells. Oncogene. 2017;36(30):4267–76.
Tabe Y, Lorenzi PL, Konopleva M. Amino acid metabolism in hematologic malignancies and the era of targeted therapy. Blood. 2019;134(13):1014–23.
Guri Y, Colombi M, Dazert E, Hindupur SK, Roszik J, Moes S, et al. mTORC2 Promotes Tumorigenesis via Lipid Synthesis. Cancer Cell. 2017;32(6):807–823.e12.
Yi J, Zhu J, Wu J, Thompson CB, Jiang X. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proceedings of the National Academy of Sciences. 2020;117(49):31189–97.
Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, et al. mTOR Complex 1 Regulates Lipin 1 Localization to Control the SREBP Pathway. Cell. 2011;146(3):408–20.
Chen Y, Qian J, He Q, Zhao H, Toral-Barza L, Shi C, et al. mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer. Oncotarget. 2016;7(18):25224–40.
Phelan JD, Young RM, Webster DE, Roulland S, Wright GW, Kasbekar M, et al. A multiprotein supercomplex controlling oncogenic signalling in lymphoma. Nature. 2018;560(7718):387–91.
Xu W, Berning P, Erdmann T, Grau M, Bettazová N, Zapukhlyak M, et al. mTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma. Leukemia. 2023;37(1):178–89.
Demicco M, Liu XZ, Leithner K, Fendt SM. Metabolic heterogeneity in cancer. Nat Metab. 2024;6(1):18–38.
Koundouros N, Poulogiannis G. Reprogramming of fatty acid metabolism in cancer. Br J Cancer. 2020;122(1):4–22.
Uddin S, Hussain AR, Ahmed M, Bu R, Ahmed SO, Ajarim D, et al. Inhibition of Fatty Acid Synthase Suppresses c-Met Receptor Kinase and Induces Apoptosis in Diffuse Large B-Cell Lymphoma. Molecular Cancer Therapeutics. 2010;9(5):1244–55.
Gifford GK, Gifford AJ, Chen Q, Shen Y, Gabrielli S, Gill AJ, et al. Fatty acid synthase and adenosine monophosphate-activated protein kinase regulate cell survival and drug sensitivity in diffuse large B-cell lymphoma. Leukemia & Lymphoma. 2020;61(8):1810–22.
Kapadia BB, Roychowdhury A, Kayastha F, Nanaji N, Gartenhaus RB. PARK2 Regulates eIF4B-Driven Lymphomagenesis. Molecular Cancer Research. 2022;20(5):735–48.
Singh AR, Gu JJ, Zhang Q, Torka P, Sundaram S, Mavis C, et al. Metformin sensitizes therapeutic agents and improves outcome in pre-clinical and clinical diffuse large B-cell lymphoma. Cancer Metab. 2020;8:10.
Smyth L, Blunt DN, Gatov E, Nagamuthu C, Croxford R, Mozessohn L, et al. Statin and cyclooxygenase-2 inhibitors improve survival in newly diagnosed diffuse large B-cell lymphoma: a large population-based study of 4913 subjects. Br J Haematol. 2020;191(3):396–404.
Wang Y, Maurer MJ, Larson MC, Allmer C, Feldman AL, Bennani NN, et al. Impact of metformin use on the outcomes of newly diagnosed diffuse large B-cell lymphoma and follicular lymphoma. Br J Haematol. 2019;186(6):820–8.

(Not answered)

Download PDF

Version 1

posted

You are reading this latest preprint version

Fatty acid metabolism provides an essential survival signal in OxPhos and BCR DLBCL cells

Status:

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

Metabolic characterization of an extended panel of DLBCL cell lines

Inhibition of FASN induces DLBCL cell death

Combining inhibition of FA metabolism with mitochondrial stress promotes DLBCL cell death

Combination of FASN and mTORC1 inhibition promotes DLBCL cell death

DISCUSSION

MATERIALS AND METHODS

Human DLBCL cell lines and culture conditions

Reagents

Measurement of intracellular ATP content

Apoptosis Assays

Cell viability assay

Lipid droplet content

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1