Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy

Abstract

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d^-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

Keywords: BRCA1; HNSCC; LRRC8A; LRRC8D; Platinum drug resistance; VRAC; breast cancer; genetically engineered mouse model; high-dose chemotherapy.

FIGURE 1

Loss of Lrrc8a or Lrrc8d induces Pt-drug resistance in BRCA1;p53-deficient mammary tumor cells. A, Representative Western blots of control (ntg B1 and ntg C1), Lrrc8a (Lrrc8a_C10 and Lrrc8a_D8), and Lrrc8d (Lrrc8d_E12 and Lrrc8d_G12) KO cell lines used in the validation experiments. B, Proliferation rates of WT and Lrrc8a or Lrrc8d KO cells. Mean ± SD of six replicates is shown. Clonogenic survival assays and quantification of WT and LRRC8A/D-deficient cell lines treated with cisplatin (C and D), carboplatin (E and F), or oxaliplatin (G and H). Representative images of selected lines and concentrations are shown. Data represent mean ± SD of three independent replicates and were fitted to a four parameter logistic sigmoidal curve. P values are calculated by one-way ANOVA followed by Tukey multiple comparisons test for the log(IC₅₀) values of the survival curves ****, P < 0.0001. I–L, Quantification of clonogenic growth assays using the different Lrrc8a (Lrrc8a_D8 + pOZ empty, + pOZ Lrrc8a wt polyclonal or clonal lines) or Lrrc8d (Lrrc8d_D8 + pOZ empty, + pOZ Lrrc8d wt polyclonal or clonal lines) rescue cell lines treated with cisplatin or carboplatin. As negative controls, empty vector-transduced cell lines were used. Data represent mean ± SD of three independent replicates and were fitted to a four parameter logistic sigmoidal curve. P values are calculated by one-way ANOVA followed by Tukey multiple comparisons test for the log(IC₅₀) values of the survival curves. ****, P < 0.0001; **, P < 0.01. M, Representative images of selected conditions of the clonogenic growth assays in the presence of cisplatin or carboplatin, using the rescue cell lines from I–L are shown.

FIGURE 2

Loss of Lrrc8a or Lrrc8d reduces cisplatin uptake and the subsequent formation of Pt-DNA adducts and DNA damage. A, CyTOF-based measurement of Pt uptake over time using 0.5 μmol/L cisplatin in nontargeting (ntg) control, Lrrc8a-, or Lrrc8d-KO cell lines. The data represent the mean ± SD of three independent replicates consisting of three technical replicates each, where approximately 100,000 cells per condition and cell lines were acquired (two-way ANOVA followed by Tukey multiple comparisons test, ****, P < 0.0001; **, P < 0.01). B, CyTOF-based measurement of Pt uptake after 24 hours treatment with 4 μmol/L carboplatin of ntg, Lrrc8a-, or Lrrc8d-KO cell lines. The data represent the mean ± SD of three independent replicates where approximately 50,000 cells per condition and cell lines were acquired (two-way ANOVA followed by Tukey multiple comparisons test, ****, P < 0.0001). C, CyTOF-based measurement of Pt uptake after 24 hours treatment with 0.5 μmol/L cisplatin, 4 μmol/L carboplatin, or 0.5 μmol/L oxaliplatin of selected clonal Lrrc8a-, or Lrrc8d-high expression rescue lines compared with the empty vector transduced KO cell line. The data represent the mean ± SD of three independent replicates where approximately 50,000 cells per condition and cell lines were acquired (two-way ANOVA followed by Tukey multiple comparisons test, ****, P < 0.0001). D, Representative images of the average nuclear staining in ntg or Lrrc8a- or Lrrc8d-KO cell lines using the NKI-A59 antibody against cisplatin-adducts in the presence or absence of 10 μmol/L cisplatin treatment for 6 hours; scale bar, 10 μm. E, Quantification of the raw integrated density per nucleus of the NKI-A59 cisplatin adduct staining, mean with ± 95% confidence interval of three independent replicates are shown. Per replicate approximately 100 nuclei were quantified. The significance was determined using two-way ANOVA followed by Tukey multiple comparison test. ****, P < 0.0001. F, Representative images of yH2AX immunofluorescence staining of Lrrc8a^−/−, Lrrc8d^−/−, and control cell lines following cisplatin treatment. Scale bar, 20 μm. G, Quantification of yH2AX foci in the nucleus of Lrrc8a^−/−, Lrrc8d^−/− and control cell lines in response to cisplatin treatment. Per cell line and condition, 200 nuclei were quantified each replicate. Median ± 95% confidence interval of three independent replicates are shown (ordinary one-way ANOVA followed by Tukey multiple comparisons test, ****, P < 0.0001).

FIGURE 3

Lrrc8a and Lrrc8d deficiency promote carboplatin resistance in vivo.A, Schematic overview of the in vivo experiment. B and C, Kaplan–Meier OS curves of mice transplanted with LRRC8A/D-deficient or ntg control tumors treated with either vehicle or 50 mg/kg carboplatin. Statistical analysis was performed with the log-rank test (Mantel–Cox). *, P < 0.05; ***, P < 0.001.

FIGURE 4

Cisplatin uptake into the kidneys of LRRC8D-deficient mice and subsequent DNA damage are reduced. A, A schematic overview of the development of Lrrc8d KO mice using CRISPR/Cas9-mediated KO of the first coding exon (2561 bp) in zygotes. B, Western blotting of LRRC8D using kidney lysates derived from WT, heterozygous and homozygous Lrrc8d KO mice. C, Representative images of the IMC tissue analysis of cisplatin- or vehicle-treated WT or LRRC8D-deficient mice. For the illustration, the most abundant isotope ¹⁹⁴Pt was used. For the visualization of the nuclei within the tissue, the signal of the iridium DNA intercalator isotope ¹⁹¹Ir is shown. Highlighted are three image sections which were used for the quantification of the mean Pt levels. Scale bar, 200 μm. D, Normalized mean ¹⁹⁴Pt kidney measurements of WT or LRRC8D deficient mice, 6 hours after treatment with 6 mg/kg cisplatin i.v. or the vehicle. Three 1 mm² sections of three kidneys per group were acquired, where three image sections from the cortical region of each acquisition were quantified. This results in 27 datapoints per group. The data represent the ¹³⁴Xe⁺ normalized mean ¹⁹⁴Pt signal of the image sections ± SD (two-way ANOVA, followed by Tukey multiple comparisons test). E, IHC using the anti-cisplatin-DNA adduct and anti-yH2AX antibodies of WT, heterozygous and homozygous Lrrc8d KO mice after the treatment with 6 mg cisplatin per kg for 6 hours. The images were taken at 40× magnification. Scale bar, 50 μm. F and G, The percentage of positive nuclei in 50 kidney cortex image sections per kidney (for NKI-A59 antibody) or the whole cortical kidney region of each mouse (for yH2AX) were quantified and normalized via the average basal levels of the untreated samples; vehicle: wt N = 5, het KO N = 8, hom KO N = 8, treated: wt N = 8, het KO N = 12, hom KO N = 16; ****, P < 0.0001. The data represent mean percentage of positive nuclei over all the image sections per mouse ± SD (two-way ANOVA, followed by Tukey multiple comparisons test). Kaplan–Meier relapse-free survival of mice transplanted with either cisplatin-naïve tumors (H) or cisplatin resistant tumors (I). The mice were treated on days 0 and 14 with the indicated dose of cisplatin (i.v.). For the treatment with 12 mg/kg i.v. the Lrrc8d KO mice were used. Statistical analysis was performed with the log-rank test (Mantel–Cox). **, P < 0.01; ***, P < 0.001.

FIGURE 5

Loss of LRRC8A or LRRC8D reduces survival parameters in patients with HNSCC treated with a chemoradiotherapy regimen. A, OS, progression, locoregional control, and distant metastasis (DM) parameters of patients with at least −1 LRRC8A copy-number loss versus control. B, Comparison of polyA RNA-seq expression data to copy-number loss of LRRC8A. Patients were classified into "low" expression group by the cutoff at around 20 rpkm. C, OS data of high (N = 129) and low (N = 58) LRRC8A-expressing patients defined by the cutoff determined in B. D, Patients were further classified into received cumulative dose groups of below or above 200 mg/m² of cisplatin. E, Correlation analysis of LRRC8A versus LRRC8D expression levels. F, OS, progression, and locoregional control parameters of patients differentially expressing LRRC8D (low N = 61, medium N = 60, high = 60). G, Distant metastasis outcome of patients with low, medium, or high LRRC8D expression. H, Distant metastasis outcome of patients differentially expressing LRRC8D further classified in high cumulative dose (left) and low cumulative dose of cisplatin (right). I, Forest plots displaying the results from CoxPH model fits for OS, PFS, locoregional control, and DM-free survival for the >200 mg/m² of cisplatin or <200 mg/m² of cisplatin treatment groups.