Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion

Abstract

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.

Keywords: autophagy; lysosome; nucleotide metabolism; pancreatic cancer; replication stress.

Fig. 1.

RSR inhibitors synergize with CQ to inhibit cell growth. (A) Composite drug interaction score of CQ and 430 kinase inhibitors screened for growth inhibition in MiaPaca2 cells (n = 2). (B) Viability of PDAC cells in 2D and 3D cultures ± CQ ± VE-822 for 72 h (n = 3). (C) Flow cytometry analyses of DNA content and DNA damage marker pH2A.X in MiaPaca2 cells treated ± CQ ± VE-822 for 24 h (n = 2). (D) The antiproliferation effect of CQ/VE-822 combination in a panel of human PDAC cell lines and primary PDAC cultures. UT, untreated. CQ: 20 µM; VE-822: 500 nM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Lysosomal inhibitors induce replication stress. (A) Time course effects of 20 μM of CQ on replication stress marker pCHEK1-S345 and on autophagy marker LC3B in MiaPaca2 cells. (B) Dose–response effects of 24-h CQ treatment on pCHEK1-S345 and LC3B in MiaPaca2 cells. (C) Effect of CQ treatment for 24 h on pCHEK1-S345 and LC3B in a panel of PDAC cell lines. (D) Effects of 24-h treatment with lysosome inhibitors on pCHEK1-S345 and LC3B in MiaPaca2. (E) Measurements of S-phase duration and G1 cell percentage by EdU pulse–chase flow cytometry analysis. All immunoblots are representative of at least two independent experiments. CQ: 20 µM unless otherwise indicated; NH₄Cl: 10 mM; BafA1: 10 nM. **P < 0.01, ***P < 0.001.

Fig. 3.

CQ treatment impairs de novo nucleotide biosynthesis. LC-MS/MS-MRM analysis of (A) relative levels of [¹³C₆]glucose-labeled dNTPs and (B) percentage of [¹³C₆]glucose labeling of the four DNA bases (DNA-C, DNA-T, DNA-A, DNA-G) in PDAC cells ± CQ (n = 3). (C) Relative levels of [¹³C₆]glucose-labeled NTPs and (D) percentage of [¹³C₆]glucose labeling of RNA in MiaPaca2 (n = 3). CQ: 20 µM; 24-h treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 4.

Asp depletion by CQ impairs de novo nucleotide biosynthesis, induces RSR, and inhibits PDAC cell proliferation. (A) Relative amino acid levels measured by LC-MS in MiaPaca2 cells ± CQ for 48 h (n = 3). (B) CQ treatment increased [¹⁴C]Asp uptake by MiaPaca2 cells. (C) [¹³C₆]glucose-labeled DNA bases (DNA-C, DNA-T, DNA-A, DNA-G) measured by LC-MS/MS-MRM in MiaPaca2 cells ± CQ ± Asp for 72 h (n = 3). (D) pCHEK1-S345 and LC3B levels in MiaPaca2 cells ± CQ ± Asp for 24 h. (E) Viability of MiaPaca2 cells ± CQ ± Asp for 72 h (n = 3). CQ: 20 µM; Asp: 10 mM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 5.

CQ causes mitochondrial dysfunction. (A) Chronic CQ treatment induces mitochondrial membrane potential heterogeneity. MTG, MitoTracker Green; TMRE, tetramethylrhodamine ethyl ester. (B) Chronic CQ treatment reduced mitochondrial respiration. OCR, oxygen consumption rate. (C) Pyruvate supplementation (1 mM) rescued CQ-induced Asp reduction in MiaPaca2 cells. (D) Pyruvate supplementation rescued CQ-induced proliferation inhibition in MiaPaca2 cells. CQ: 20 µM; acute: 2 h; chronic: 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 6.

CQ and VE-822 synergistically inhibit tumor cell growth in organotypic in vitro and in vivo PDAC models. (A) Viability of MiaPaca2-GFP cells in CAF coculture ± CQ ± VE-822 (n = 3). (B) Representative fluorescence images of A. (Scale bar, 0.05 μm.) (C) Xenograft MiaPaca2 tumor growth ± CQ ± VE-822. (D) IHC staining of indicated markers in MiaPaca2 tumors after 5-d vehicle or CQ/VE-822 treatments. (E) Kaplan–Meier curves of tumors in syngeneic KPC mouse models ± CQ ± VE-822. (F) IHC staining of indicated markers in KPC tumors with 5-d vehicle or CQ/VE-822 treatments. *P < 0.05, **P < 0.01, ***P < 0.001.