Fig 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).

Fig 3: 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.

Fig 4: 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.