Loss of UXS1 Selectively Depletes Pyrimidines and Induces Replication Stress in KEAP1-Mutant Lung Cancer

Abstract

Kelch-like ECH-associated protein 1 (KEAP1) is the third most commonly mutated gene in non-small cell lung cancer and is associated with poor prognosis. In this study, we investigated synthetic lethal interaction genes in KEAP1-mutated cancer cells and identified a dependency on UDP-xylose synthase 1 (UXS1), which converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose in the proteoglycan synthetic pathway. UDP-glucose dehydrogenase (UGDH), a transcriptional target of NRF2 that converts UDP-glucose to UDP-GlcA, was highly expressed in KEAP1-mutant tumors. Upon UXS1 knockdown, depletion of UDP-xylose occurred in both KEAP1-mutant and wild-type cells, whereas UDP-GlcA accumulated to a greater extent in the KEAP1-mutant setting. The resulting shortage of available UDP and other pyrimidines slowed S-phase progression and stalled DNA replication fork marks, causing cells to undergo prolonged cell-cycle exit or apoptosis. Dependency on UXS1 was rescued by knocking out UGDH to prevent UDP-GlcA accumulation and UDP depletion. DNA replication stress in UXS1-depleted cells sensitized them to clinical cell-cycle checkpoint inhibitors. Furthermore, CRISPR screening experiments identified genes that modulate UXS1 dependency. Whereas the liver had the highest normal tissue expression of UGDH, UXS1 knockout in the liver did not result in hepatotoxicity. Taken together, these data demonstrate that UXS1 is a selective dependency in KEAP1-mutant tumors, and loss of UXS1 creates additional therapeutically exploitable vulnerabilities in KEAP1-mutant tumors.

Significance: UXS1 loss in KEAP1-mutant cells causes pyrimidine nucleotide depletion, DNA replication stress induction, and ultimately cell-cycle exit that results in tumor stasis, highlighting UXS1 as a potential therapeutic target in KEAP1-mutant tumors. See related commentary by Yasseen and DeNicola, p. 4582.

Graphical abstract

Figure 1.

KEAP1-mutant NSCLC cells are sensitive to UXS1 loss in a UGDH expression–dependent manner. A, Dependency on the indicated genes (data from DepMap) of all cell lines harboring either KEAP1-WT or KEAP1-mutant status. B, Effects of UXS1 knockdown in KEAP1-mutant and -WT cell lines. C, Time-course of cell count following CRISPR gene KOs in a KEAP1-mutant cell line. PLK1 KO is used as a positive control for loss of proliferation. D and E, Xenograft tumor growth of H2122 and A549 cell lines expressing dox-inducible shRNA against UXS1 or a nontargeting control (NTC). F, Gene dependency data of UXS1 KO versus UGDH expression for all cell lines (data from DepMap). G, UGDH expression of KEAP1-WT or -mutant lung lines, color coded for UXS1 gene effect. H, Western blot of UGDH expression in the indicated cell lines. β-Actin is shown as a loading control, and quantitation of UGDH band intensities is shown below. I, KEAP1-mutant cells are sensitive to UXS1 knockdown, which is lost if UGDH is knocked out. P values were generated by unpaired t test. J, KEAP1-WT cells are sensitized to UXS1 loss by transient overexpression (OE) of UGDH. P values were generated by unpaired t test. K, Diagram showing the metabolic pathways involving UGDH and UXS1. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant. TPM, transcripts per million.

Figure 2.

Loss of UXS1 enzymatic activity results in hyperaccumulation of UDP-GlcA and a loss of pyrimidine nucleotides. A, Time-course metabolic profiling of UDP–xylose and UDP-GlcA in A549 KEAP1-mutant cells expressing shNTC versus shUXS1. B, Metabolic profiling of UDP–xylose and UDP-GlcA in H460 and H2122 KEAP1-mutant cells after shUXS1 induction or UGDH KO. C and D, Targeted metabolomic profiling of H2122 and H460 KEAP1-mutant lung cancer cells after shUXS1 induction. As expected UDP-GlcA is the most increased metabolite. Notably, pyrimidine nucleotides (bold font) are significantly depleted. Right, metabolomic profiling after UGDH KO in the same cell lines, in which UXS1 loss no longer causes significant metabolic changes. E, Time-course metabolomics of A549 KEAP1-mutant cells showing the temporal dynamics of pyrimidine, but not purine, nucleotide loss. F, Rescue of pyrimidine nucleotides by uridine (100 µmol/L) or cytidine (100 µmol/L) supplementation after 6 days of shUXS1 induction in H2122 cells. G, Diagram of the metabolic process blocked by targeting UXS1, resulting in insufficient UDP recycling. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant.

Figure 3.

UXS1 loss in KEAP1-mutant cancer cells causes DNA replication stress. A, Pathway analysis of significantly altered genes from RNA-seq data in KEAP1-mutant lines after indicated times of shUXS1 induction. Left, downregulated pathways show significant loss of cell-cycle signaling and translational processes. Right, upregulated pathways show increased DNA damage signaling and p53-driven apoptotic pathways. B, DNA synthesis rate measured by EdU incorporation in KEAP1-mutant or -WT cells after 6 or 12 days of shRNA induction. C, Introduction of a live-cell biosensor for Geminin abundance, a metric of S and G₂ cell-cycle phases. Time-lapse imaging of this sensor after 4 days of dox pretreatment (shUXS1 induction) shows that KEAP1-mutant cells spend significantly longer durations in S/G₂. D, Immunofluorescence imaging of FANCD2, a protein that forms distinct nuclear foci at sites of stalled replication forks. Images and quantification demonstrate that shUXS1 causes a time-dependent significant increase in these DNA replication stress lesions, which can be rescued by supplementation of free pyrimidine nucleosides uridine (100 µmol/L) or cytidine (100 µmol/L). Turbo-RFP images are shown as a positive control for shRNA induction. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, nonsignificant.

Figure 4.

DNA replication stress resulting from UXS1 loss leads to states of cell-cycle exit and apoptosis. A, Illustration of movement through cell states related to cell-cycle status and cell death. B, Quantitation of apoptotic cell states in the indicated cell lines after 0, 6, or 12 days of shUXS1 induction, measured by flow cytometry. Different states and corresponding quadrant gating are shown in Supplementary Fig. S4. C, Fate mapping after UXS1 loss across the indicated cell lines and treatment durations of dox. Pie charts are constructed by first bifurcating populations into viable or dead, using the flow cytometry apoptosis data. The viable cells are then stratified further by phospho-Rb status, as quantified by immunofluorescence imaging in Supplementary Fig. S4. D, Percentage of cells staining positive for SA-β-gal after the indicated treatments and times. Generated P value is from an unpaired t test. Uridine was used at 100 µmol/L. Palbociclib (1 µmol/L) was used as a positive control for generating senescent cells. E, Immunofluorescence imaging quantification of p21 protein after shUXS1 induction alone or in combination with uridine supplementation (100 µmol/L). F, Imaging and automated nuclei counting of H2122 cells after 7 days of shUXS1 induction alone or in combination with uridine (100 µmol/L) or cytidine (100 µmol/L) supplementation. **, P ≤ 0.01; ns, nonsignificant.

Figure 5.

CRISPR KO screening reveals additional effectors of sensitivity to UXS1 loss. A, Summary volcano plot of CRISPR KO screen in H460 and H2122 cells, plotted as the shUXS1 condition relative to shNTC induction after 11-day treatments. Positive and negative significance cutoff values for each axis are marked by the shaded boxes. B, Venn diagram of resistance gene hits for each cell line, in which the overlapping region between the two cell lines is displayed as an expanded table. UGDH marks an expected hit, and SLC35E3 and TP53 are highlighted as hits of interest based on their mechanistic role in nucleotide sugar transport and proliferation control, respectively. C, Validation experiments after KO of TGDS or SLC35E3 in H2122 cells and assessment of relative viability after 7 days of shUXS1 induction. Generated P values by unpaired t test. D, Validation experiments after KO of TGDS or SLC35E3 in H2122 cells and assessment of UDP-GlcA and UDP abundance after 4 days of shUXS1 induction. Generated P value summaries are from unpaired t tests. E, DepMap KO data of cell lines harboring either no TP53 mutations or at least one TP53 hotspot mutation. All cell lines are shown on the left, and UGDH^high lines are shown on the right. UGDH^high lines are defined as those with UGDH expression of log₂ (TPM+1) > 6 in the 24Q2 Public dataset. TPM, transcripts per million. F and G, Pathway analysis obtained through Metascape, in which the inputs were the CRISPR KO screen hits that conferred sensitivity, from A. Top 15 pathway hits are displayed. Common hits relate to cell-cycle progression, highlighted in red text. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant.

Figure 6.

UXS1 loss sensitizes KEAP1-mutant cells to cell-cycle checkpoint kinase inhibitors. A, Illustrated mechanism of DNA replication stress signaling after UXS1 loss. B, Single-cell quantification of fluorescent imaging for EdU versus pCDK2 or pCDK1, inhibitory phosphorylations made by checkpoint kinases. Cells cycling through shUXS1 induction experience increases in these replication stress marks. WEE1 inhibitor (adavoserib, 250 nmol/L) was used as a positive control to reduce these phosphospecies. C and D, Imaging and single-cell quantification of γH2AX after UXS1 loss, alone or in combination with PKMYT1i (lunresertib, 500 nmol/L) or WEE1i (250 nmol/L). E, Flow cytometric measurement of apoptotic cells (Annexin V–positive) after indicated treatments in H2023 cells. Generated P value summaries are from unpaired t tests. F and G, Dose–response experiments combined with dox-inducible shUXS1. UXS1 loss offers more complete response to these kinase inhibitors and to 5-fluorouracil (5-FU) at a later time point (ATR inhibitor used was ceralasertib). H, Time-course combinations across multiple KEAP1-mutant or -WT cells. Responder lines experience cooperative killing from the combinations tested. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 7.

UXS1 loss is well tolerated by normal mouse livers, a tissue with high UGDH expression. A, Schematic of LNP delivery for mouse liver assessments. IV, intravenous. B, Western blots of liver tissue lysates after targeted KOs via LNP delivery. EGFP is used as a delivery control. Ugt1a1 KO is used as a positive control for loss of glucuronidation in downstream assays. C, Body weight measurements over the duration of the in vivo LNP experiment. Mean and SEM of six mice plotted per time point. D, Measurement of liver enzymes at indicated times of the in vivo LNP experiment. Mean and SEM of six mice plotted per time point. E and F, Metabolite measurements for each tumor sample, presented as boxplots showing the full range and individual samples. Generated P values by unpaired t test. Bilirubin buildup is measured as a metabolite of hepatotoxicity, seen in the positive control Ugt1a1 KO but not in Uxs1 KO; P values are generated by unpaired t test. ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant. ALKP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transferase.