Oncogenic KRAS Regulates Amino Acid Homeostasis and Asparagine Biosynthesis via ATF4 and Alters Sensitivity to L-Asparaginase

Abstract

KRAS is a regulator of the nutrient stress response in non-small-cell lung cancer (NSCLC). Induction of the ATF4 pathway during nutrient depletion requires AKT and NRF2 downstream of KRAS. The tumor suppressor KEAP1 strongly influences the outcome of activation of this pathway during nutrient stress; loss of KEAP1 in KRAS mutant cells leads to apoptosis. Through ATF4 regulation, KRAS alters amino acid uptake and asparagine biosynthesis. The ATF4 target asparagine synthetase (ASNS) contributes to apoptotic suppression, protein biosynthesis, and mTORC1 activation. Inhibition of AKT suppressed ASNS expression and, combined with depletion of extracellular asparagine, decreased tumor growth. Therefore, KRAS is important for the cellular response to nutrient stress, and ASNS represents a promising therapeutic target in KRAS mutant NSCLC.

Keywords: ASNS; ATF4; KEAP1; KRAS; L-asparaginase; NRF2; PI3K; asparagine; glutamine; synthetic vulnerability.

Figure 1. Response to KRAS knockdown is influenced by nutrient status

(A) Western blot of cell lines expressing doxycycyline-inducible shGFP or shKRAS cultured with 1μg/mL doxycycline for indicated number of days. (B) Western blot of indicated cell lines expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hours. (C, D, E) Heatmap of differentially expressed genes for cell lines cultured in (C) 4mM or 0.5mM glutamine for 72 hours (D) cells expressing shGFP or shKRAS cultured in 4mM glutamine or (E) cells expressing shGFP or shKRAS cultured in 0.5mM glutamine for 72 hours. (F) Venn diagram of genes differentially regulated by glutamine deprivation, or by KRAS knockdown in either 4mM or 0.5mM glutamine. (G) Comparison of directionality of expression changes after glutamine deprivation or KRAS knockdown in 0.5mM glutamine. (H) Pathways differentially regulated in 0.5mM glutamine, by KRAS in 0.5mM glutamine, or by both. (I, J) Analysis of overall survival for (I) total cohort of NSCLC patients or (J) the subset with confirmed KRAS mutation with patients were stratified into high or low expression of the 100 genes regulated by both glutamine deprivation and by KRAS in 0.5mM glutamine. Human lung cancer gene expression data were from the TCGA cohort (http://cancergenome.nih.gov/). Corresponding mutation data for same samples were downloaded from the Xena browser (https://xenabrowser.net/) (see STAR Methods). See also Figure S1.

Figure 2. KRAS cooperates with the GCN2-p-eIF2 pathway to induce ATF4

(A) Transcription factors with enriched binding sites in genes differentially regulated by both glutamine deprivation and by KRAS in 0.5mM glutamine. Heat map scales are ranked by −log (p-value). Weighted binding motifs were analyzed and acquired using Transfac 2016.1. (B) RT-qPCR for indicated ATF4 target genes (results are the average of 3 technical replicates) and (C) Western blots for cell lines expressing shGFP or shKRAS in 4mM or 0.5mM glutamine for 72 hours. (D) Western blots of cells expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine in the presence of actinomycin D (1ng/mL) or cycloheximide (10μg/mL) where indicated for 12 hrs. (E) RT-qPCR for ATF4 in indicated NSCLC cells expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hrs (results are the average of 3 technical replicates). (F) RT-qPCR (results are the average of 3 technical replicates) for indicated cells or (G) Western blots for for H2009 cells expressing shGFP or shGCN2 cultured in 4 or 0.5mM glutamine for 72 hrs. Error bars represent mean +/− SEM. See also Figure S2.

Figure 3. NRF2 is required for KRAS-mediated regulation of ATF4 via PI3K

(A) RT-qPCR (results are the average of 3 technical replicates) and (B) Western blots from cells not-treated (NT), or treated with 5μM BKM120, 5μM MK2206, 10μM AZD6244, 100nM Rapamycin or 10μM BRD7389 for 72 hrs, with glutamine withdrawal for the last 36 hours. (C) RT-qPCR (results are the average of 3 technical replicates) and (D) Western blots for A549 cells expressing shGFP or shKRAS and constitutively active myr-AKT or kinase dead myr-K179M AKT cultured in 4mM or 0.5mM glutamine for 72 hrs. (E) Western blots for NRF2 for experiment described in (B). (F) Western blots of indicated cells expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hrs. (G) RT-qPCR (results are the average of 3 technical replicates) and (H) Western blots of H2009 and A549 cells expressing shGFP or shNRF2 cultured in 4mM or 0.5mM glutamine for 72 hrs. (I) RT-qPCR (results are the average of 3 technical replicates) for indicated cells expressing shGFP or shNRF2 at indicated time-points (in hours) after glutamine withdrawal. Protein levels of (J) KEAP1 or (K) NRF2 from lung tumors with altered or unaltered expression of ATF4 (see Figure S3B) Human lung cancer gene expression and protein data were obtained from the TCGA network (http://cancergenome.nih.gov/). Error bars represent mean +/−SEM. See also Figure S3.

Figure 4. The role of ATF4 is dependent on nutrient status and genetic context

(A) Viability of indicated NSCLC cell lines expressing shATF4 relative shGFP grown in the indicated glutamine concentration for 48 hours (results are the average of 3 technical replicates). (B) Western blots of indicated cells cultured cultured in 0.5mM glutamine for 72 hrs. (C) Western blots of indicated cells expressing shGFP or shATF4 cultured in 4 mM or 0.5mM glutamine for 72 hrs. (D) RT-qPCR for ATF4 targets from cells with wild-type (wt) KRAS and wt KEAP1, an oncogenic mutation (mut) in KRAS and wt KEAP1, or KRAS mut and KEAP1 mut cultured in indicated glutamine concentrations relative to cells cultured in 4mM glutamine (results are the average of 3 technical replicates). (E) Viability of cell lines bearing the indicated mutations cultured in 0, 0.25, or 0.5 mM glutamine for 48 hrs relative to 4mM glutamine. Each data point represents the average of 3 technical replicates from one cell line. (F) RT-qPCR (results are the average of 3 technical replicates) and (G) Western blots of H358 cells expressing shGFP or shKEAP1 cultured in 4mM or 0.1mM glutamine for 72 hrs. (H) Growth curves, and (I) final tumor weights of H460 xenograft tumors with CRISP-Cas9 mediated deletion of ATF4 (sgGFP n=7; sgATF4.6 n=4; sgATF4.8 n=7). Error bars represent mean +/−SEM; p-values were calculated by a two-tailed t-test. See also Figure S4.

Figure 5. KRAS regulates amino acid transport and metabolism during nutrient stress

(A) RT-qPCR (results are the average of 3 technical replicates) and (B) Western blots for amino acid transporters in cell lines expressing dox-inducible shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hrs. (C) ³H-Glutamine and (D) ³H-leucine uptake measured by scintillation count in A549 or H460 cell lines expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hrs (normalized to cell number). Results are the average of three biological replicates. (E) Intracellular amino acid levels in H460 cells expressing shGFP or shKRAS cultured in 4mM or 0.5mM glutamine for 72 hrs (normalized to cell number). Results are the average of 2 biological replicates. Error bars represent mean +/−SEM; p-values were calculated by a two-tailed t-test. See also Figure S5.

Figure 6. KRAS contributes to protein biosynthesis and supports mTORC1 signaling via regulation of ASNS

(A) Western blots of indicated NSCLC cells cultured in 4mM or 0.5mM glutamine for 72 hrs with addition of 0.5mM asparagine where indicated. (B) Western blots of H460 cells expressing pQB-GFP or pQB-ASNS cultured in indicated concentration of glutamine for 72 hrs. (C) Western blots of indicated NSCLC cells expressing shGFP or shASNS cultured in 4mM glutamine with addition of 0.5mM asparagine where indicated for 72 hrs. (D) Growth curves of A549 cells expressing shGFP or shATF4 cultured in 4mM glutamine DMEM with or without asparagine (Asn) for 72 hrs or with overexpression of ASNS (pQB-ASNS). Results are average of 3 biological replicates. (E, F) Protein synthesis rates (incorporation of ³H-Leucine into peptides) for H460 cells expressing shGFP or shKRAS cultured in 4mM glutamine with (E) addition of 500μM asparagine where indicated or (F) over-expression of ASNS. Results are average of 3 biological replicates. (G) Protein synthesis rates (incorporation of ³⁵S-Methionine) for H460 cells expressing shGFP or shKRAS cultured in 4mM glutamine with addition of 500μM asparagine where indicated for 96 hours. Results are average of 3 biological replicates. (H) Western blots of H460 cells expressing shGFP or shASNS cultured in 4mM or 0.5mM glutamine for 72 hrs with addition of 0.5mM asparagine where indicated. (I) Western blots of H460 and A549 cells expressing shGFP or shKRAS in 4mM or 0.5mM glutamine for 72 hrs. (J) Western blots of H460 and A549 cells expressing shGFP or shKRAS in 4mM or 0.5mM glutamine for 72 hrs with addition of 0.5mM asparagine where indicated. Error bars represent mean +/− SEM. p-values were calculated by a two-tailed t-test; *p<0.05, **p<0.001. See also Figure S6.

Figure 7. Asparagine synthetase is rate-limiting for tumorigenesis

(A) Western blots of ASNS in lung tumors dissected from KRAS-LSL^G12D;p53^fl/fl mice 12 weeks after adenoviral Cre administration. (B) Growth curves, and (C) final tumor weights of A549 xenograft tumors expressing pQB-GFP (n=5) or pQB-ASNS (n=5). (D) Western blots of tumors from (B, C). (E) Growth curves and (F) final tumor weights of H460 xenograft tumors with CRISPR-Cas9 mediated deletion of ASNS treated with vehicle or L-asparaginase (L-Asp) (sgGFP+Vehicle n=7; sgGFP+L-Asp n=11; sgASNS5.3+Vehicle n=5; shASNS5.3+L-Asp n=8; sgASNS5.7+Veh n=5; shASNS5.7+L-Asp n=7). (G) Growth curves and (H) final tumor weights of H460 xenograft tumors treated with vehicle (n=7), L-asparaginase (3UI/kg daily) (n=4), MK2206 (120 mg/kg 3X/week) (n=7) or both (n=5). (I) RT-qPCR and (J) Western blots of tumors dissected from (G, H) for ASNS levels. Error bars represent mean +/− SEM. p-values were calculated by a two-tailed t-test: *p<0.05, **p<0.001. See also Figure S7.

Figure 8. Mechanisms involved in regulation of amino acid metabolism by oncogenic KRAS

(Left) KRAS enhances ATF4 mRNA levels through PI3K-AKT mediated NRF2 up-regulation. ATF4 translation is stimulated during nutrient deprivation by activation of the GCN2-p-eIF2 pathway, resulting in up-regulation of ATF4 protein levels, and ATF4 target genes. ASNS is an ATF4 target gene responsible for asparagine biosynthesis, which contribute to protein synthesis, suppression of apoptosis and up-regulation of mTORC1. (Right) When AKT is inhibited, abrogating ATF4 up-regulation during nutrient deprivation, and extracellular asparagine is depleted by the chemotherapeutic, L-asparagine, cells are asparagine starved, and tumor growth is suppressed.