Translational reprogramming marks adaptation to asparagine restriction in cancer

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Abstract

While amino acid restriction remains an attractive strategy for cancer therapy, metabolic adaptations limit its effectiveness. Here we demonstrate a role of translational reprogramming in the survival of asparagine-restricted cancer cells. Asparagine limitation in melanoma and pancreatic cancer cells activates receptor tyrosine kinase–MAPK signalling as part of a feedforward mechanism involving mammalian target of rapamycin complex 1 (mTORC1)-dependent increase in MAPK-interacting kinase 1 (MNK1) and eukaryotic translation initiation factor 4E (eIF4E), resulting in enhanced translation of activating transcription factor 4 (ATF4) mRNA. MAPK inhibition attenuates translational induction of ATF4 and the expression of its target asparagine synthetase (ASNS), sensitizing melanoma and pancreatic tumours to asparagine restriction, reflected in inhibition of their growth. Correspondingly, low ASNS expression is among the top predictors of response to inhibitors of MAPK signalling in patients with melanoma and is associated with favourable prognosis when combined with low MAPK signalling activity. These studies reveal an axis of adaptation to asparagine deprivation and present a rationale for clinical evaluation of MAPK inhibitors in combination with asparagine restriction approaches.

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Fig. 1: ATF4 activity impedes growth suppression in response to asparagine limitation.
Fig. 2: In silico pan-tumour analysis predicts RTKs, BRAF, and MNK1 as ASNS SL partners.
Fig. 3: The MAPK pathway is required for ATF4 upregulation on asparagine limitation.
Fig. 4: ATF4 induction following ASNS suppression requires MAPK–mTORC1–eIF4E signalling.
Fig. 5: MNK1 is essential for ATF4 induction in response to asparagine limitation.
Fig. 6: ASNS suppression upregulates RTKs to induce MAPK signalling.
Fig. 7: Combined l-Aase treatment and MEK inhibition suppresses pancreatic and melanoma tumour growth.

Data availability

The pancancer data were retrieved from TCGA Research Network: http://cancergenome.nih.gov/. The dataset used to infer the SL interactions in the earlier stage of the study is available in Broad GDAC Firehose (https://gdac.broadinstitute.org). For other TCGA analysis which was performed in the later stage, we used more updated datasets that are available through UCSC Xena Browser (https://xenabrowser.net). In particular, https://xenabrowser.net/datapages/?cohort=TCGA%20Pan-Cancer%20(PANCAN) and https://xenabrowser.net/datapages/?cohort=TCGA%20TARGET%20GTEx. All patients’ data were analyzed from published papers that are referenced and publicly available accordingly. Raw data for the GC-MS figures were deposited in Figshare with the Digital ObjectIdentifier 10.6084/m9.figshare.9887984. All data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank D. Tuveson and members of his laboratory for providing KPC cells, M. Herlyn for providing the melanoma cell lines, members of the L. Larue laboratory for establishing the NRAS mutant melanoma cell line MaNRAS1 (1007), M. Leibovitch for technical assistance, O. Zagnitko for metabolic analyses and members of the Ronai lab for continued discussion. The Cancer Metabolism Core at Sanford Burnham Prebys Medical Discovery Institute is supported by National Cancer Institute Cancer Center Support Grant P30CA030199. I.T. is a scholar of the Fonds de Recherche du Québec-Santé (FRQS; Junior 2). Support from National Cancer Institute grants R35CA197465, P01CA128814 and Hevery Foundation Gift (to Z.A.R.) are gratefully acknowledged.

Author information

G.P. and Z.A.R. conceived the study; G.P., E.H., I.T., E.R. and Z.A.R. designed experiments; G.P., E.H., K.T., S.V. and Y.F. performed experiments; G.P. and D.A.S. conducted metabolic analyses; L.L. provided valuable reagents; G.P., J.S.L., A.D.S., E.H., K.T., E.R. and Z.A.R. analysed data; and G.P., E.R., I.T. and Z.A.R. wrote the manuscript.

Correspondence to Gaurav Pathria or Ze’ev A. Ronai.

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Competing interests

Z.R. is co-founder and serves as scientific advisor to Pangea Therapeutics. E.R. was co-founder (divested) and serves as a non-paid scientific advisor to Pangea Therapeutics.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 ASNS Suppression Induces the Amino Acid Response Pathway.

a, GC-MS-based estimation of intracellular asparagine levels in A375 and UACC-903 cells 72 hr after treatment with si-ASNS. b, Relative proliferation of A375 and UACC-903 cells treated with si-ASNS#1 or si-ASNS#2, with or without supplementation with L-asparagine (L-Asn; 0.3 mM), over the indicated time course. c, Immunoblotting of ASNS, ATF4, phosphorylated and total GCN2 and eIF2α in melanoma cells 72 hr after treatment with si-ASNS, L-Asn (0.3 mM), or both. d, Immunoblotting of ATF4 in melanoma lines 72 hr after treatment with si-ASNS, ISRIB, or both (left). Relative proliferation of melanoma cells treated as in (left) for indicated time course (right). Data are representative of three independent experiments and presented as mean ±SEM of n=3 biological replicates in b and d. Data shown as mean ±SEM of n=3 independent experiments in a. Statistical significance was calculated using two-tailed unpaired Student’s t-test for a and two-way Anova for b and d. In b, black and orange P values correspond to the comparison between si-ASNS#1 and si-ASNS#1+L-Asn and si-ASNS#2 and si-ASNS#2+L-Asn respectively. Source Data

Extended Data Fig. 2 In-Silico Pan-Tumor Analysis Predicts Synthetic Lethal Partners of ASNS.

a, Sensitivity to the PLK1/3 inhibitor GW843682X of pan-tumor cell lines segregated based on high (ASNShi) and low (ASNSlo) ASNS expression. P-value calculated by one-sided Wilcoxon rank sum test. Each dot represents a cell line (ASNShi, n=204; ASNSlo, n=20). Middle line and the whiskers represent the mean and the standard deviation respectively. b, Conditional essentiality of PLK1 (ASNShi, n=24; ASNSlo, n=22), JAK3 (ASNShi, n=69; ASNSlo, n=67) and ATF4 (ASNShi, n=69; ASNSlo, n=67) in ASNSlo and ASNShi cell lines. One-sided Wilcoxon rank sum P values are denoted for each gene knockdown. Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile, respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range, and the top and bottom whiskers represent the maximum and the minimum values respectively.

Extended Data Fig. 3 MAPK Signaling is Critical for ATF4 Induction Upon Asparagine Limitation.

a, Proliferation of melanoma cells as measured 72 hr after indicated treatment. Proliferation is shown relative to mock (NT-siRNA and DMSO)-treated cells, set to 1.0. b, Immunoblotting of indicated proteins in melanoma cells 72 hr after indicated treatment. c, qRT-PCR analysis of indicated transcripts in melanoma cells 48 hr after treatment with si-ASNS, PLX-4032, or both. d, Immunoblotting of indicated proteins 72 hr after treatment with si-ASNS, PD-325901 or both. e, Immunoblotting of phosphorylated and total ERK1/2 protein in indicated cancer cell lines 72 hr after combined treatment with si-ASNS and L-Asn, with or without L-A’ase. f, Immunoblotting of phosphorylated ERK1/2 in melanoma cells 72 hr after treatment with si-ASNS#1 or #2, with or without supplementation with L-Asn. g, Proliferation of cancer cell lines measured 72 hr after treatment with si-ASNS, PD-325901, or both relative to mock. h, GC-MS-based estimation of intracellular Ser, Gly, and Ala levels in UACC-903 cells 72 hr after treatment with si-ASNS. i, qRT-PCR analysis of indicated transcripts in UACC-903 cells 48 hr after treatment with si-ASNS, PLX-4032, or both. j, GC-MS-based 13C6-glucose fractional isotope labeling of serine and glycine in UACC-903 cells 72 hr after treatment with si-ASNS, PLX-4032, or both. k, qRT-PCR analysis of LDHA transcript in UACC-903 cells 48 hr after indicated treatment. Data are representative of three independent experiments and presented as the mean ±SEM of n=3 biological replicates in a and g, mean ±SEM of n=3 technical replicates in c and k, mean ±SEM of n=2 technical replicates in i. Data shown as mean ±SEM of n=3 independent experiments in h and j. Statistical significance was calculated using two-tailed unpaired Student’s t-test, except in a, where ordinary one-way Anova was used. Source Data

Extended Data Fig. 4 The MAPK-mTORC1-eIF4E Axis is Essential for ATF4 Induction Following Asparagine Limitation.

a, Immunoblotting of phosphorylated and total S6 protein in melanoma cells 72 hr after treatment with si-ASNS, si-ATF4, si-BRAF, or indicated combinations. Fig. 3d and Extended Data Fig. 4a show parts of the same experiment and share the internal control (HSP90). b, Immunoblotting of phosphorylated and total S6 protein and 4E-BP1 in melanoma cells 72 hr after treatment with si-ASNS, SCH-772984, or both. c, Immunoblotting of ATF4 and phosphorylated and total S6 protein in Mia-Paca-2 cells 72 hr after treatment with si-ASNS#1 or #2, PD-325901, or a combination of the respective si-ASNS and PD-325901. d, Immunoblotting of ATF4 and phosphorylated and total S6 protein in pancreatic cancer cell lines 72 hr after treatment with si-ASNS, Rapamycin (Rapa), or both. e, Immunoblotting of ATF4 and phosphorylated and total S6 protein in Mia-Paca-2 cells 72 hr after treatment with si-ASNS, Torin 1, or both. f, Proliferation of cancer cell lines measured 72 hr after treatment with si-ASNS, Rapa, or both. Values are shown relative to mock (NT-siRNA and DMSO)-treated cells. Data are representative of three independent experiments and presented as the mean ±SEM of n=3 biological replicates in f. Statistical significance was calculated using two-tailed unpaired Student’s t-test. Source Data

Extended Data Fig. 5 MNK1 is Critical for ASNS Suppression-Associated ATF4 Induction.

a, Immunoblotting of MNK1 and ATF4 in melanoma cells 72 hr after treatment with si-ASNS, si-MNK1#1, #2 or #3, or indicated combinations of si-ASNS with si-MNK1#1-#3. b, Immunoblotting of ATF4 and MNK1 in Mia-Paca-2 and Panc-1 cells 72 hr after treatment with si-ASNS, si-MNK1, or both. c, Proliferation of cancer cell lines 72 hr after treatment with si-ASNS, si-MNK1, or both relative to NT-siRNA treated cells (set to 1.0). d, qRT-PCR analysis of MNK1 transcript in melanoma cells 48 hr after treatment with indicated si-ASNS. e, Immunoblotting of phospho and total MNK1 and eIF4E proteins in melanoma cells after 72 hr treatment with si-ASNS and L-Asn (0.3 mM), with or without L-A’ase. f, qRT-PCR analysis of eIF4E transcript in melanoma cells 48 hr after treatment with si-ASNS. g, qRT-PCR analysis of H3A and CDH1 mRNA levels in subpolysomal, light, and heavy polysomal fractions of A375 cells treated for 48 hr with si-ASNS relative to mock treatment, set to 1.0. h, Immunoblotting of ATF4 and phospho and total eIF4E protein in UACC-903 cells treated with si-ASNS, eFT508, or both for 72 hr. i and j, Immunoblotting of phospho and total MNK1 and eIF4E proteins in melanoma cells treated 72 hr with si-ASNS, SCH-772984, or both (i), or si-ASNS, Rapa, or both (j). Data are representative of three independent experiments and presented as the mean ±SEM of n=3 biological replicates in c, mean ±SEM of n=4 technical replicates in d and g, and mean ±SEM of n=3 technical replicates in f. Statistical significance was calculated using two-tailed unpaired Student’s t-test. Source Data

Extended Data Fig. 6 Asparagine Limitation Increases RTK Expression.

a, Immunoblotting of EGFR in melanoma cells 72 hr after treatment with indicated si-ASNS. b, qRT-PCR analysis of EGFR transcript in melanoma cells 48 hr after treatment with si-ASNS. c-e, Immunoblotting of EGFR in melanoma cells 72 hr after treatment either individually or as a combination with si-ASNS and Torin 1 (c), si-ASNS and si-eIF4E (d), or si-ASNS and si-MNK1 (e). f, Immunoblotting of phospho and total ERK1/2 in melanoma cells treated 72 hr with si-ASNS, EGFR inhibitor (Gefitinib), or both. g, qRT-PCR analysis of VEGFR-2 transcript levels in melanoma cells 48 hr after treatment with si-ASNS#2 or #3. h, (left) Immunoblotting of VEGFR-2, PDGFR-B, α-Tubulin, and GAPDH in melanoma cells treated with NT-siRNA, si-ASNS#1 or #2 followed by L-azidohomoalanine (AHA) labeling (see Methods). (right) Ponseau-S staining of streptavidin pull-down fraction from melanoma cells left untreated or treated with AHA. i, Immunoblotting of VEGFR-2 and PDGFR-B in melanoma cells 72 hr after treatment with si-ASNS, ISRIB, or both. j, Immunoblotting of the indicated proteins in melanoma cells 72 hr after treatment with si-ASNS, si-GCN2, or both. Data are representative of three independent experiments and presented as the mean ±SEM of n=3 technical replicates in b and g. Source Data

Extended Data Fig. 7 L-Asparaginase and MEK Inhibitor Combination Suppresses In Vivo Tumor Growth.

a, Comparison of ASNS expression in cancer vs. healthy tissue samples from TCGA and GTEx dataset using two-sided Wilcoxon rank sum test. b, Proliferation of melanoma and pancreatic cancer cell lines as measured 72 hr after treatment with L-A’ase, PD-325901, or both relative to mock-treated cells. All cell lines were grown in L-Asn-supplemented DMEM. c, Volume of tumours from C3H/HeN mice injected subcutaneously with SW1 mouse melanoma cells and treated as indicated (n=8 mice per group). Statistical analysis: Welch’s t-test (two-tailed). In c, brown, blue, and pink P values correspond to the comparison between L-A’ase+PD-325901 (2.5 mg/Kg) and Vehicle, L-A’ase+PD-325901 (2.5 mg/Kg) and L-A’ase, and L-A’ase+PD-325901(2.5 mg/Kg) and PD-325901 (2.5 mg/Kg) respectively. In c, green, red, and black P values correspond to the comparison between L-A’ase+PD-325901(5.0 mg/Kg) and Vehicle, L-A’ase+PD-325901 (5.0 mg/Kg) and L-A’ase, and L-A’ase+PD-325901(5.0 mg/Kg) and PD-325901 (5.0 mg/Kg) respectively. In a, LUSC, Lung Squamous Cell Carcinoma; DLBC, Diffuse Large B-Cell Lymphoma; COAD, Colon Adenocarcinoma; ESCA, Esophageal Carcinoma; SARC, Sarcoma; UCEC, Uterine Corpus Endometrial Carcinoma; OV, Ovarian Cancer; KICH, Kidney Chromophobe; BLCA, Bladder Urothelial Carcinoma; CESC, Cervical Squamous Cell Carcinoma; KIRP, Kidney Renal Papillary Cell Carcinoma; BRCA, Breast Cancer; HNSC, Head and Neck Squamous Cell Carcinoma; KIRC, Kidney Renal Clear Cell Carcinoma; LUAD, Lung Adenocarcinoma; LIHC, Liver Hepatocellular Carcinoma; TCGT, Tenosynovial Giant Cell Tumor; LAML, Acute Myeloid Leukaemia; PRAD, Prostate Adenocarcinoma; GBM, Glioblastoma Multiforme; STAD, Stomach Adenocarcinoma; THCA, Thyroid Carcinoma; LGG, Low Grade Glioma. Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile, respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range, and the top and bottom whiskers represent the maximum and the minimum values respectively. In b, data are representative of three independent experiments and presented as the mean ±SEM of n=4 biological replicates and the statistical significance was calculated using two-tailed unpaired Student’s t-test. Source Data

Supplementary information

Reporting Summary

Supplementary Table 1

ASNS Synthetic Lethal Partners.

Supplementary Table 2

Gene List- Predictors of Response to MAPK Signaling Inhibition.

Supplementary Table 3

qRT-PCR Primer List.

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Source Data Extended Data Fig. 6

Unprocessed Western Blots

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Statistical Source Data

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Pathria, G., Lee, J.S., Hasnis, E. et al. Translational reprogramming marks adaptation to asparagine restriction in cancer. Nat Cell Biol 21, 1590–1603 (2019) doi:10.1038/s41556-019-0415-1

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