The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but minimal efficacy with substantial toxicity in clinical trials. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, these findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

. Trx1 and TrxR1 is highly expressed in NSCLC tumor samples and is associated with poor prognosis. Figure S1A. Expression profile of Trx1 in NSCLC subtypes lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) that represent different datasets obtained from the Oncomine database. P value was determined by a two-tailed Mann-Whitney test. Figure S1B. Expression profile of TrxR1 in LUAD and LUSC that represents different datasets obtained from the Oncomine database. P value was determined by a two-tailed Mann-Whitney test. Figure S1C. Expression of Trx1 and TrxR1 in LUAD (N=560; Trx1 p<.00001; TrxR1 p<.00001) and LUSC (N=546; Trx1 <.00001; TrxR1 <.00001), compared to normal tissue. The expression of both Trx1 and TrxR1 in more than 50% of NSCLC patients are higher than the 75% quantile expression level the normal samples. The data is from TCGA. Figure S1D. Survival curves (Kaplan-Meier) and the association of higher Trx1 and TrxR1 expression with poor overall survival in patients with LUAD (Trx1; **** p <0.0001; TrxR1; ** p =0.0017). The data is from TCGA. [Statistical information: n=3, All histograms depict mean value; error bars represent ± SD. The pvalues were calculated using one way ANOVA for multiple comparison; ns: non-significant] K.
(B) Representative western blots of the DNA-binding protein RPA32 and phosphorylated RPA32(p-RPA32 S33 and S4/8) and γH2AX in H1299 cells with Trx1 or TrxR1 depletion. Cytoplasmic and chromatin fractions were fractionated using mild and stringent detergent-based buffers respectively.
(C) An illustration of the scheme to measure nuclear intensity of RS markers.
(G and H) Representative immunofluorescence images of p-RPA32(S33) and γH2AX staining in the indicated groups. Bar graphs shows the frequency of cells with ≥5 foci/cell in the indicated groups.
(I and J) The degree of accumulation of cells in the S phase upon Trx1 or TrxR1 depletion by flow cytometric profile of cell cycle progression in the indicated groups (I) and its quantitation (J).

(K) Representative western blots of RS-related proteins in untransformed human normal epithelia BEAS cells upon Trx1 or TrxR1 depletion.
[Statistical information: n=3, All histograms depict mean value; error bars represent ± SD. The pvalues were calculated using one way ANOVA for multiple comparison. Red line in dot plot indicates mean; ** p≤0.005; *** p ≤0.001; **** p ≤0.0001].  [Statistical information: n=3, All histograms depict mean value; error bars represent ± SD. The pvalues were calculated using one way ANOVA for multiple comparison; *p≤0.05; ** p≤0.005] Figure S6. Figure S6. Levels of the indicated dNTPs in cells with Trx1 or TrxR1 depletion with or without in combination of CHK1 inhibition. Total cellular dNTPs were subjected to RT-based primer extension assay to quantify each dCTP, dGTP and dTTP concentration.
[Statistical information: n=3 All histograms depict mean value; error bars represent ± SD. The pvalues were calculated using one way ANOVA for multiple comparison; *p≤0.05; ** p≤0.005]