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Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma

Abstract

Alterations in components of the SWI/SNF chromatin-remodeling complex occur in ~20% of all human cancers. For example, ARID1A is mutated in up to 62% of ovarian clear cell carcinomas (OCCC), a disease lacking effective therapies. Here we show that ARID1A mutation creates a dependence on glutamine metabolism. SWI/SNF represses glutaminase (GLS1) and ARID1A inactivation upregulates GLS1. ARID1A inactivation increases glutamine utilization and metabolism through the tricarboxylic acid cycle to support aspartate synthesis. Indeed, glutaminase inhibitor CB-839 suppresses the growth of ARID1A mutant, but not wild-type, OCCCs in both orthotopic and patient-derived xenografts. In addition, glutaminase inhibitor CB-839 synergizes with immune checkpoint blockade anti-PD-L1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation. Our data indicate that pharmacological inhibition of glutaminase alone or in combination with immune checkpoint blockade represents an effective therapeutic strategy for cancers involving alterations in the SWI/SNF complex, such as ARID1A mutations.

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Fig. 1: ARID1A inactivation creates a dependence on glutamine.
Fig. 2: GLS1 is a direct target of the SWI/SNF complex.
Fig. 3: Inactivation of the SWI/SNF complex sensitizes cells to GLS inhibition.
Fig. 4: ARID1A inactivation increases glutamine-dependent aspartate biosynthesis.
Fig. 5: GLS inhibition suppresses growth of ARID1A-inactivated OCCCs in vivo.
Fig. 6: GLS inhibition in combination with immune checkpoint blockade suppresses the growth of Arid1a/Pik3ca OCCC.

Data availability

The previously published ChIP-seq data that were re-analyzed here are available in the Gene Expression Omnibus under accession codes GSE120060 (ref. 45), GSE69566 (ref. 46), GSE124225 (ref. 47) and GSE123284 (ref. 48). Previously published RNA-seq data that were re-analyzed here are available under accession codes GSE106665 (ref. 49) and GSE124227 (ref. 47). Previously published ATAC-seq data that were re-analyzed here are available under accession codes GSE124224 (ref. 47), GSE106665 (ref. 49) and GSE101966 (ref. 50). Metabolomics data have been deposited into MassIVE under accession code MSV000086347. Cancer Cell Line Encyclopedia RNA-seq data were downloaded from https://portals.broadinstitute.org/ccle/data/. Human lung adenocarcinoma, renal clear cell carcinoma, skin cutaneous melanoma and uterine corpus endometrial carcinoma data were derived from https://www.cbioportal.org/. Source data for unprocessed immunoblots for Figs. 1c, 2b,e–j and 3a,f and Extended Data Figs. 1a,b, 2f,h and 3f and source data used for statistical analyses have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Z. Stine and C. Simon from the University of Pennsylvania for UMRC2 and RCC4 cell lines. This work was supported by US National Institutes of Health grants (R01CA160331, R01CA163377, R01CA202919, R01CA239128 and P01AG031862 to R. Z.; P50CA228991 to R. Z. and R.D.; K99CA241395 to S.K.; R50CA221838 to H.-Y.T., S10OD023658 and S10OD023586 to D.W.S.; R01CA195670 to D.G.H., F31CA247336 to J.Z. and T32CA009191 to T.N.) and US Department of Defense (OC180109 and OC190181 to R. Z.). The Honorable Tina Brozman Foundation for Ovarian Cancer Research and the Tina Brozman Ovarian Cancer Research Consortium 2.0 (to R.Z. and R.D.) and Ovarian Cancer Research Alliance (Collaborative Research Development Grant no. 596552 to R.Z. and Ann and Sol Schreiber Mentored Investigator Awards no. 598026 to S.W. and no. 649658 to J.L.). Support of Core Facilities was provided by Cancer Centre Support Grant CA010815 to The Wistar Institute.

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S.W., T.F., J.L., T.N., Y.W., D.O., H.L., N.F., J.A.Z., S.K., W.Z., H.-Y. T. and Z.T.S. performed the experiments and analyzed data. S.W., C.V.D. and R.Z. designed the experiments. Q.L. and A.V.K. performed statistical analysis. L.E.S. and R.D. contributed key experimental materials. D.H., D.W.S. and R.Z. supervised studies. S.W, Y.W., D.W.S., Z.T.S., C.V.D. and R.Z. wrote the manuscript. R.Z. conceived the study.

Corresponding author

Correspondence to Rugang Zhang.

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Peer review information Nature Cancer thanks Steven de Jong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ARID1A inactivation creates a dependence on glutamine.

a-b, Validation of ARID1A knockout in parental and ARID1A knockout RMG1 (a) and OVCA429 (b) cells. Immunoblots are representative of three independent experiments with similar results. c, Top 10 metabolic pathways enriched by ARID1A knockout in OVCA429 cells determined by metabolites set enrichment analysis (MSEA). d, Contribution of glutamine to oxygen consumption in the indicated OVCA429 cells expressing shARID1A or control analyzed by Seahorse. n= 5 independent experiments. e, Colony formation and quantification of parental and ARID1A knockout OVCA429 cells with or without glutamine deprivation for 12 days. n= 4 independent experiments. f-g, A fluorescence glucose analog 2-NBDG-based glucose uptake assayed by flow cytometry analysis for the indicated parental and ARID1A knockout RMG1 (f) or OVCA429 (g) cells. n= 4 independent experiments. h, Colony formation of parental and ARID1A knockout RMG1 cells cultured in the medium with indicated concentration of glucose for 12 days. Shown are representative images of four independent experiments. Error bars represent mean with s.d. in d, e, f and g. P values were calculated using two-tailed Student t-test in d, e and Fisher’s least significant difference test in c.

Source data

Extended Data Fig. 2 GLS1 is a direct target of the SWI/SNF complex.

a, Expression of glutamine metabolism related genes in control and ARID1A knockout RMG1 cells determined by RNA-seq analysis. Note that GLS1 shows the highest upregulation in response to ARID1A knockout. n=3 independent experiments. b, The indicated ChIP-seq and input tracks in the GLS1 gene locus in parental and ARID1A knockout RMG1 cells in previously published datasets (GSE120060). c, The indicated ChIP-seq and input tracks in the GLS1 gene locus in the indicated cancer cells based on the public database mining (GSE69566, GSE124225, GSE123284 and GSE106665). d, ATAC-seq tracks in the GLS1 gene locus in parental and ARID1A knockout cells based on the indicated datasets (GSE124224, GSE106665 and GSE101966). e, Expression of GLS1 mRNA in the indicated cancer cells based on based on mining public databases (GSE124227 and GSE106665). f-g, Control and ARID1A knockdown OVCA429 cells were examined for expression of ARID1A and GLS1 by immunoblot (f) or measured for glutaminase activity (g). n= 4 independent experiments. h, Control and ARID1A knockout ES2 cells were examined for expression of ARID1A and GLS1 by immunoblot. i, The association of ARID1A, BAF155, SNF5 and RNA Pol II with the GLS1 gene promoter in parental and ARID1A knockdown OVCA429 cells was examined by ChIP-qPCR analysis. An isotype matched IgG was used as a control. n = 3 independent experiments. j, Control and wildtype ARID1A ectopically expressing OVISE cells were examined for expression of ARID1A and GLS1 by immunoblot. k, Inverse correlation between GLS1 and ARID1A expression in 274 TP53 wildtype cancer cell lines across cancer types in the Cancer Cell Line Encyclopedia RNAseq database. Immunoblots are representative of three independent experiments with similar results in f, h and j. Error bars represent mean with s.d. in e, g and i. P value was calculated using two-tailed Student t-test in e, g, i and Spearman correlation analysis in k.

Source data

Extended Data Fig. 3 Inactivation of SWI/SNF complex sensitizes cells to glutaminase inhibition.

a, Validation of GLS1 knockdown by qRT-PCR in parental and ARID1A knockout RMG1 cells expressing the indicated shGLS1s or control. n= 3 independent experiments. b, Colony formation by the indicated cells treated with the indicated doses of CB-839. Shown are representative images of 4 independent experiments with similar results. c, Dose response curves to glutaminase inhibitor CB-839 determined by colony formation assay in the indicated ARID1A-mutated OCCC and VHL-deficient renal clear cell carcinoma (RCC) cell lines. n=4 independent experiments. d, Differential sensitivity of TP53 wildtype cell lines for the indicated cancer types with SWI/SNF wildtype or mutation to GLS1 knockdown in the Project Achilles dataset. Specifically, GLS1 shRNA sensitivity score for 384 cell lines along with mutation status of member of SWI/SNF complex (ARID1A, ARID1B, SMARCA2, PHF10, SMARCA4, SMARCB1, SMARCC1, SMARCC2, SMARCD3, DPF2, ACTL6A) and TP53 were downloaded from Broad Cancer Cell Line Encyclopedia database. Only 118 cells lines with wildtype TP53 were taken for analysis. Cell lines were grouped by source tissue site and categorized into mutant (at least one mutation in any members of SWI/SNF complex) and wildtype SWI/SNF complex groups. Average sensitivity scores to GLS1 RNAi for each tissue and SWI/SNF complex groups were calculated. Average mutant SNI/SNF scores were plotted versus difference between mutant and wildtype SWI/SNF complex on a bubble plot to illustrate cancer types with association between GLS1 RNAi and SWI/SNF mutation. Size of the data circles were proportional to the number of cells lines in the tissue group. Note that the criteria for including in the analysis is with minimal 5 cell lines in the database. e, Sensitivity score of SWI/SNF wildtype or mutated skin cancer cell lines with wildtype TP53 to GLS1 knockdown in the Project Achilles dataset. f-g, Expression of GLS1 in control and GLS1 ectopically expressed ARID1A wildtype RMG1 cells determined by immunoblot (f). And the indicated cells were subjected to dose response curves to glutaminase inhibitor CB-839 determined by colony formation assay (g). n=4 independent experiments. Immunoblots are representative of two independent experiments with similar results in f. Error bars represent mean with s.d. in a, c, e and g. P values were calculated using two-tailed Student t-test in a, e and one-tailed Student t-test in d.

Source data

Extended Data Fig. 4 ARID1A inactivation increases glutamine-dependent aspartate biosynthesis.

a, Quantification the indicated metabolites determined by glutamine tracing in control and ARID1A knockout RMG1 cells. n= 3 independent experiments. b, Quantification of colony formation by parental or ARID1A knockout OVCAR429 cells cultured in medium supplemented with or without 5 mM aspartate treated with or without CB-839 (0.1 μM or 0.25 μM). n= 4 independent experiments. c, Expression of SLC1A3 in RMG1 and OVCA429 cells determined by qRT-PCR analysis. n= 3 independent experiments. d, Expression of SLC1A3 in ARID1A knockout RMG1 cells with or without ectopic SLC1A3 expression determined by qRT-PCR analysis. n= 3 independent experiments. e, Cell cycle distribution in RMG1 ARID1A KO cells treated with or without 1 μM CB-839 for 72 hrs determined by flow cytometry analysis. n= 3 independent experiments. f, Schematic of glutamine-dependent aspartate biogenesis through the TCA cycle. g-h, Relative expression of genes encoding for enzymes that contribute to aspartate biogenesis from glutamine through the TCA cycle determined by qRT-PCR analysis in parental control and ARID1A knockout RMG1 cells (g) or OVCA429 cells with or without ARID1A knockdown (h) cells. Validation of 3 independent experiments as shown in Extended Data Fig. 2a. Error bars represent mean with s.d. in a, b, c, d, e, g and h. P values were calculated using two-tailed Student t-test in a, b, c, d, e, g and h.

Source data

Extended Data Fig. 5 Glutaminase inhibitor CB-839 suppresses the growth of ARID1A-inactivated OCCCs in vivo.

a, Schematic of experimental design and reference time of the mouse experiment. Cells were orthotopically transplanted into non-obese diabetic/severe combined immunodeficiency gamma (NSG) mice and allowed to establish for one week. After the tumors presented palpable masses, the mice were randomized into various treatment groups and treated for an additional three weeks. At the end of treatment of three weeks, mice from various treatment groups were euthanized for measuring tumor weight as a surrogate for tumor burden or followed for survival experiment. b-c, Orthotopic xenografts formed by ARID1A knockout (b) or control RMG1 cells (c) were treated with vehicle or CB-839 for 3 weeks (n=7 mice/group). At the end of the treatment, tumor weight was measured as surrogate for tumor burden. d-e, Tumors dissected from b-c, were subjected to immunological staining for GLS1, cell proliferation marker Ki67, mitotic marker serine 10 phosphorylated histone H3 (pH3S10) or apoptosis marker cleaved caspase 3 on serial sections (d) and the histological score (H-score) of the indicated markers was quantified from three separate fields from seven tumors from seven individual mice in each of the indicated treatment groups (e). Scale bar = 100 μm. f, Orthotopic xenografts formed by ARID1A-mutated TOV21G cells were treated with vehicle or CB-839 for three weeks (n=6 mice/group). Body weight of tumor bearing mice was measured at the indicated time point. Error bars represent mean with s.d. in b, c, e and f. P values were calculated using two-tailed Student t-test in b, c and e.

Source data

Extended Data Fig. 6 CB-839 does not affect PDL1 expression.

a, The gating strategy used for determining the percentage of PD1+/CD8+ T cell populations. b, ARID1A-mutated TOV21G cells were treated with vehicle or CB-839 (100 nM) for 48 hours and expression of PDL1 was examined by flow cytometry analysis. n = 3 independent experiments. Error bars represent mean with s.d. in b. P values were calculated using two-tailed Student t-test in b.

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Wu, S., Fukumoto, T., Lin, J. et al. Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma. Nat Cancer 2, 189–200 (2021). https://doi.org/10.1038/s43018-020-00160-x

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