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cGAS–STING drives the IL-6-dependent survival of chromosomally instable cancers

Abstract

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS–STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6–STAT3-mediated signalling, which depends on the cGAS–STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS–STING signalling and explains why the cGAS–STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

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Fig. 1: Loss of cGAS–STING-mediated signalling impairs the proliferation of TNBC cells that display CIN.
Fig. 2: IL-6–IL-6R–STAT3 signalling induced by cGAS and STING is required for survival of TNBC cells undergoing CIN.
Fig. 3: Inhibition of IL-6R impairs the growth of CIN-induced TNBC tumours.
Fig. 4: IL-6 signalling is required for cancer cells that display acute or chronic CIN in vitro and in vivo observed across cancer types.

Data availability

All data from this study are available by request by contacting F.F. The RNA sequencing data have been deposited into ArrayExpress under the accession number E-MTAB-10923. Shallow single-cell whole-genome sequencing data have been deposited into the European Nucleotide Archive under the accession number PRJEB49800.  Source data are provided with this paper.

Code availability

Analysis code is available at https://github.com/mschubert/cgas_ko.

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Acknowledgements

We are grateful to the members of the Bruggeman, de Bruyn, van Vugt and Foijer laboratories for fruitful discussions. We thank M. Weij and D. Eichorn at the central animal facility for assistance with animal experiments; J. Teunis at the flow cytometry facility for help with flow cytometry; R. Arjaans and N. Halsema at the UMCG/ERIBA Research Sequencing facility for help with RNA sequencing library preparation and (single cell) sequencing; and M. Broekhuis and J. Seiler at the UMCG/ERIBA iPSC/CRISPR facility for advice regarding the CRISPR knockout experiments. This work was supported by Dutch Cancer Society grants to F.F. (2015-RUG-7833 & 2018-RUG-11457) and M.A.T.M.v.V. (2018-RUG-11352), UMCG research fellowships to C.H. and M. Requesens, and a UMCG Cancer Research Fund (KRF) grant to C.H.

Author information

Authors and Affiliations

Authors

Contributions

C.H., M.d.B., M.A.T.M.v.V. and F.F. conceived the project. C.H. and F.F. designed all the experiments. C.H., A.E.T., A.v.d.B., L.A.R., P.L.B. and T.v.d.S. performed the in vitro experiments. C.H., A.E.T. and M. Requesens performed the animal experiments. M.S., M. Roorda, M.C. and R.W. performed data analyses. W.P. contributed reagents. D.C.J.S. oversaw RNA and single-cell sequencing. B.v.d.V. oversaw histological staining. C.H. and F.F. wrote the manuscript with input from M.S., M.d.B. and M.A.T.M.v.V. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Marco de Bruyn, Marcel A. T. M. van Vugt or Floris Foijer.

Ethics declarations

Competing interests

M.A.T.M.v.V. has acted on the Scientific Advisory Board of Repare Therapeutics, which is unrelated to this work. B.v.d.V has acted as a consultant or scientific advisory board member (on request) for Visiopharm, Philips, MSD/Merck and as a speaker for Visiopharm, Diaceutics and MSD/Merck for which UMCG was compensated. This is all unrelated to this work. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Inhibition of Mps1 and Wee1 lead to activation of cGAS signalling in BT549 TNBC cells.

a) Quantification of mitotic abnormalities observed in BT549 cells expressing Histone H2B-mCherry treated with DMSO, 250 nM reversine or 500 nM AZD1775 assessed by live cell imaging, the number of assessed mitotic cells – n - is indicated in the figure. Dashed lines indicate that these measurements (DMSO and reversine-treated) cells are also shown in Fig. 1b. b) Representative immunofluorescence images of DMSO-, 250 nM reversine-, or 500 nM AZD1775-treated BT549 cells stained with DAPI (DNA) and an anti-cGAS antibody, scale bar equals to 10 µm. Arrows point at cGAS positive micronuclei. c) Quantification of cGAS positive micronuclei in DMSO-, 250 nM reversine, or 500 nM AZD1775-treated BT549 cells. Error bars represent the s.e.m, significance was tested with a two-sided t-test. d) Representative images of 250 nM reversine- or 500 nM AZD1775-treated BT549 cells expressing Histone H2B-GFP and CENPB-mCherry, scale bar equals to 5 µm. Arrow indicates CENPB positive micronuclei. e) Quantification of CENPB positive micronuclei observed in 250 nM reversine- or 500 nM AZD1775-treated BT549 cells, the number of quantified cells – n – is indicated in the figure. f) DNA fibre assay showing decreased fibre size for AZD1775-, but not reversine-treated cells indicating replication stress in the former. BT549 cells were treated with DMSO, 250 nM reversine, or 500 nM AZD1775. For each condition, 260 fibres from 2 experiments were analysed and individual IdU track lengths are displayed together with the mean and standard deviation value. g) cGAMP levels quantified in cell lysates (intracellular) or in harvested culture media (extracellular) from wild type BT549 cells incubated with DMSO, 250 nM reversine, or 500 nM AZD1775 for 48 hours. cGAMP levels were normalised to harvested cell numbers. h) Representative images of 250 nM reversine- or DMSO-treated BT549 cells stained with DAPI (DNA) and an anti-STING antibody showing peri-nuclear STING localization following reversine treatment. Scale bar equals to 5 µm. i) Quantification of cells with perinuclear or non-perinuclear STING localization as shown in (h). The number of quantified cells – n – is indicated in the figure. j) Immunoblots on lysates from 48 hours DMSO, 250 nM reversine or 500 nM AZD1775-treated BT549 cells showing phosphorylated and total IRF3, STAT1 and STAT3. β-Actin is shown as a loading control. Error bars in (c, g) represent s.e.m, n=3 biological replicates (c, g), significance was tested with a two-sided t-test.

Source data

Extended Data Fig. 2 Loss of cGAS or STING in BT549 TNBC cells results in increased sensitivity to Mps1 or Wee1 inhibition.

a) Immunoblots of wild type and cGASKO BT549 cell lysates stained with anti-cGAS and anti-ß-actin antibodies. ß-Actin is used as a loading control. b) cGAMP levels quantified in cell lysates (intracellular) or in tissue culture media (extracellular) isolated from cGASKO BT549 cells incubated with DMSO, 250 nM reversine, or 500 nM AZD1775 for 48 hours. cGAMP levels were normalised to harvested cell numbers. c) Representative images of 250 nM reversine- or DMSO-treated BT549 cGASKO cells stained with DAPI (DNA) and an anti-STING antibody. Scale bar equals 5 µm. d) Quantification of cells with peri-nuclear or non-peri-nuclear STING localization. The number of quantified cells – n – is indicated in the figure. e) Immunoblots of cell lysates from wild type, cGASKO, STINGKO, and cGAS; STINGDKO BT549 cells treated with DMSO or 250 nM reversine stained with anti-p-IRF3 (Serine 396), anti-IRF3, anti-p-STAT1 (Tyrosine 701), anti-STAT1, anti-p-STAT3 (Tyrosine 705), anti-STAT3, or anti-ß-Actin. ß-Actin is used as a loading control. f) DMSO-normalised cell count of wild type or cGASKO BT549 cells treated with 500 nM AZD1775 for 48 hours. g) Percentage of apoptotic cells quantified by Annexin V staining of wild type or cGASKO BT549 cells treated with 500 nM AZD1775 for 48 hours. h) DMSO-normalised cell count of wild type BT549 cells treated with a combination of 2.5 μM RU.521 and 250 nM reversine for 72 hours. i) DMSO-normalised cell count of wild type BT549 cells treated with a combination of 2.5 μM RU.521 and 500 nM AZD1775 for 48 hours. j) Quantification of mitotic abnormalities observed in cGASKO BT549 cells expressing Histone H2B-mCherry treated with DMSO, 250 nM reversine or 500 nM AZD1775 assessed by live cell imaging, the number of assessed mitotic cells – n –is indicated in the figure. k) Immunoblots of wild type, STINGKO, or cGAS; STINGDKO BT549 cell lysates stained using anti-STING, anti-cGAS and anti-β Actin antibodies. ß-Actin is used as a loading control. l) Control DMSO-normalised cell counts of wild type, cGASKO, STINGKO, or cGAS; STINGDKO BT549 cells treated with 500 nM AZD1775 for 48 hours. Error bars in (b, f–i, l) represent the s.e.m, n=3 biological replicates (b, g–i, l), n=6 biological replicates (f), significance was tested with a two-sided t-test.

Source data

Extended Data Fig. 3 Loss of cGas signalling sensitizes mouse 4T1 TNBC cells to acute and chronic CIN in cell cultures and in vivo.

a) Immunoblots of wild type, cGasKO and STINGKO 4T1 cell lysates to validate loss of cGas (top panel) and STING (bottom panel) protein. ß-Actin is used as a loading control. b) Representative images of DMSO- or 250 nM reversine-treated wild type or cGasKO 4T1 cells stained with DAPI (DNA) and anti-STING antibody showing reduced peri-nuclear STING localization in cGasKO 4T1 cells. Scale bar equals to 5 µm. c) Quantification of cells with peri-nuclear and non-perinuclear localization of STING. The number of quantified cells – n – is shown in the figure. d) Quantification of mitotic abnormalities observed in wild type or cGasKO 4T1 cells expressing Histone H2B-mCherry treated with DMSO, 250 nM reversine, or 500 nM AZD1775. e–f) Control DMSO-normalised cell counts for wild type or cGasKO 4T1 cells treated with 250 nM reversine for 72 hours (e) or 500 nM AZD1775 for 48 hours (f). g–h) Fraction of apoptotic cells quantified by Annexin V staining of wild type or cGasKO 4T1 cells treated with 250 nM reversine for 72 hours (g) or 500 nM AZD1775 for 48 hours (h). i) Quantification of mitotic abnormalities observed in wild type; cGasKO, or STINGKO 4T1 cells expressing KIF2C (CINlow) or dnMCAK (CINhigh). j) Immunoblots of lysates from wild type, cGasKO, or STINGKO KIF2C (CINlow) or dnMCAK (CINhigh) expressing 4T1 cells for phosphorylated and total IRF3, STAT1 and STAT3. ß-Actin is used as a loading control. k) KIF2C-normalised cell counts for wild type, cGasKO, or STINGKO dnMCAK expressing 4T1 cells. l) Tumour-free survival of KIF2C (CINlow) or dnMCAK (CINhigh) wild type, cGasKO, or STINGKO cells transplanted to immunocompetent Balb/c and followed for up to 22 days. Tumours were included in the survival curve if they reached a volume of 500 mm3 or larger. m) End mass of tumours harvested shown in Fig. 1f and Extended Data Fig. 3l. Error bars in (e–h, k–m) indicate the s.e.m, n=5 biological replicates (e), n= 6 biological replicates (f), n=3 biological replicates (g–h, K), n=8 biological replicates (m), significance was tested with a two-sided t-test.

Source data

Extended Data Fig. 4 Effect of cGAS on differential expression of reversine-treated BT549 cells.

a) Differential expression of a parental BT549 cell line upon 48h of treatment with reversine vs. DMSO. X-axis shows log2 fold change, y-axis FDR-adjusted p-values. Size of the circle corresponds to the expression level in the untreated condition. Biological processes are characterised as MSigDB Hallmark (middle) and DoRothEA transcription factor target genes (right). Significance of gene sets was tested comparing the Wald statistic of genes within vs. genes outside the set using a linear model. Size of the circle corresponds to the number of genes in the set. b) Differential expression of cGASKO BT549 cells reversine vs. DMSO shows a clearly diminished transcriptional response, indicating that most of the transcriptional response to CIN is driven by cGAS, characterised by a drop of interferon response genes (middle) and STAT1/NF-kB-responsive genes. c) Differential expression of reversine-treated BT549 cells between cGASKO and wild type (parental) confirms a lower expression of interferon and STAT1-driven genes in the knockout vs. the wild type condition.

Extended Data Fig. 5 Acute and chronic CIN activate STAT1 and STAT3 signalling with STAT1 promoting and STAT3 preventing CIN-induced loss of viability in BT549 and 4T1 TNBC cells.

a) Control DMSO-normalised CCL5 and CXCL10 expression in 250 nM reversine-treated wild type, cGASKO, STINGKO, cGAS; STINGDKO, STAT1KO, cGAS; STAT1DKO, STAT3KO, or cGAS; STAT3DKO BT549 cells determined by quantitative RT-PCR. b) Control KIF2C-normalised Ccl5 and Cxcl10 expression in dnMCAK expressing wild type, cGasKO, and STINGKO 4T1 cells determined by quantitative RT-PCR. c) Immunoblots of wild type, STAT1KO, or cGAS; STAT1DKO BT549 cell lysates showing STAT1 or cGAS expression (top panels) and of wild type, STAT1KO and STAT1; STAT3DKO cell lysates showing STAT3 expression. ß-Actin is shown as a loading control. d) Immunoblots of wild type, STAT3KO, or cGAS; STAT3DKO BT549 cell lysates showing STAT1 or cGAS expression. ß-Actin is shown as a loading control. e) Control DMSO-normalised cell counts for wild type, cGASKO, STAT1KO, cGAS ;STAT1DKO, STAT3KO, and cGAS; STAT3DKO BT549 cells treated with 500 nM AZD1775 for 48 hours. f) Relative cell counts of doxycycline-inducible KIF2C- (CINlow) or dnMCAK- (CINhigh) expressing wild type, cGASKO, STAT1KO, cGAS; STAT1DKO, STAT3KO or cGAS; STAT3DKO BT549 cells. g) DMSO-normalised cell viability of wildtype, cGASKO or STAT3KO BT549 cells expressing KIF2C or dnMCAK across a range of UMK-57 concentrations. h) Relative expression of doxycycline-induced STAT1 (mutants) compared to endogenous STAT1 transcripts determined by quantitative RT PCR. Cells were treated with 100 ng/ml and 500 ng/m of doxycycline as indicated. i) DMSO-normalised cell counts of wild type, cGASKO, or STAT3KO BT549 cells with overexpression of wild type STAT1, constitutive active STAT1Y701E, or inactive STAT1Y701A. Error bars in (a–b, e–f, g, i) represent the s.e.m, while those in (h) represent the standard deviation. n=3 biological replicates (a–b, e–f, g, i) or n=3 technical replicates (h). Significance was tested by two-sided t-test. Cells were treated for 48 hours (a) or 72 hours (e–i). * P<0.05, ** P<0.01, *** P<0.0005 (g), exact P-values are specified in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 BT549 cells rely on non-canonical NF-kB signalling, but not canonical NF-kB signalling for survival to prevent CIN-induced cell death.

a) Immunoblots of wild type, RelAKO, or cGAS; RelADKO BT549 cell lysates showing RelA and cGAS protein levels. ß-Actin is used as a loading control. b) Immunoblots of wild type, RelBKO, or cGAS; RelBDKO BT549 cell lysates showing RelB and cGAS protein levels. ß-Actin is used as a loading control. c) Control DMSO-normalised cell counts of wild type, cGASKO, RelAKO, or cGAS; RelADKO, or RelBKO BT549 cells treated with 500 nM AZD1775 for 48 hours. d) Immunoblots of wild type, NIKKO, cGAS; NIKDKO BT549 cell lysates showing NIK and cGAS protein levels. ß-Actin is used as a loading control. e–f) Control DMSO-normalised cell viability of wild type, cGASKO, NIKKO, or cGAS; NIKDKO for increasing reversine (e) or AZD1775 concentrations (f), treated for 72 or 48 hours, respectively. g) Immunoblots of wild type, TRAF2KO, and cGAS; TRAF2DKO BT549 cell lysates showing TRAF2 and cGAS protein levels. ß-Actin is used as a loading control. h–i) Control DMSO-normalised cell viability of wild type, cGASKO, TRAF2KO, or cGAS; TRAF2DKO for increasing reversine (h) or AZD1775 concentrations (i), treated for 72 or 48 hours, respectively. j) Control DMSO-normalised cell viability of DMSO- or 250 nM reversine-treated wild type or cGASKO BT549 cells across a range of ASK1 inhibitor concentrations. k–l) Control DMSO-normalised cell viability of DMSO- or 500 nM AZD1775-treated wild type or cGASKO BT549 cells across a range of (k) ASK1 inhibitor or (l) JNK inhibitor concentrations. Bars in (c, e–f, h–i) represent the s.e.m, n=4 biological replicates (c), n=3 biological replicates (e–f, h–i). Significance was tested with a two-sided t-test. *P<0.05, **P<0.01, ***P<0.005, exact P-values are specified in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 IL6 is induced in a cGAS-STING dependent manner following drug-induced CIN in TNBC cells to promote cell survival.

a) Control KIF2C-normalised IL6 expression in dnMCAK expressing wildtype, cGASKO and STINGKO 4T1 cells determined by quantitative RT-PCR. b) Cell viability of DMSO- or 500 nM AZD1775-treated wild type or cGASKO BT549 cells supplemented with increasing quantities of IL6. c) Cell viability of DMSO- or 10 μM 5-fluoro-uracil (5-FU)-treated wild type BT549 cells supplemented with increasing quantities of IL6 to show that IL6 does not increase viability for a compound that does not provoke CIN. d–e) Control DMSO-normalised cell viability of wild type or cGASKO BT549 cells with or without overexpression of IL6 with increasing reversine (d) or AZD1775 concentrations (e). f) Control DMSO-normalised cell viability of DMSO- and 250 nM reversine-treated wild type or cGasKO 4T1 cells supplemented with increasing quantities of IL6. g) Control DMSO-normalised cell viability of DMSO- or 500 nM AZD1775-treated wild type or cGasKO 4T1 cells supplemented with increasing quantities of IL6. h–j) Control DMSO-normalised cell viability of DMSO- or 500 nM AZD1775-treated wild type, cGASKO, STINGKO, and cGAS; STINGDKO BT549 cells (h) wild type, cGASKO, STAT3KO, or cGAS; STAT3DKO BT549 cells (i), and wild type or RelBKO BT549 cells (j) supplemented with increasing quantities of IL6. k) Control DMSO-normalised cell viability of DMSO- or 250 nM reversine-treated wild type or cGASKO BT549 cells supplemented with increasing quantities of IFNa2. l) Control DMSO-normalised cell viability of DMSO- or 500 nM AZD1775-treated wild type or cGASKO BT549 cells supplemented with increasing quantities of IFNa2. m) Control DMSO-normalised cell viability of DMSO- or 250 nM reversine-treated wild type, cGASKO, STAT3KO, or cGAS; STAT3DKO BT549 cells supplemented with increasing quantities of IFNa2. n) Control DMSO-normalised cell viability of DMSO- or 500 nM AZD1775- treated wild type, cGASKO, STAT3KO, or cGAS; STAT3DKO BT549 cells supplemented with increasing quantities of IFNa2. Grey lines in (i, m–n) represent replotted data from previous panels to simplify comparison. Error bars in all panels represent the s.e.m, n=3 biological replicates (a, c–e, g–j, m–n) n=4 biological replicates (b, k–l), n=5 biological replicates (f). Significance was tested with a two-sided t-test. *P<0.05, **P<0.01, ***P<0.005, exact P-values are specified in Source Data Extended Data Fig. 7. Cells were treated for 48 hours (b, e, g–j, l, m) or 72 hours (c, d, f, k, m).

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Extended Data Fig. 8 Tocilizumab treatment selectively targets TNBC cells but not untransformed mammary cells displaying CIN.

a) Quantification of mitotic abnormalities observed in MDAMB231, MDAMB436, E0771 or MCF10A cells expressing Histone H2B-cherry treated with DMSO or 250 nM reversine. b–e) Control DMSO-normalised cell viability of DMSO- or 250 nM reversine-treated MDAMB231(b), MDAMB436 (c), E0771 (d) and MCF10A (e) cells treated with increasing concentrations of tocilizumab. f–g) Quantification of mitotic abnormalities observed in Histone H2B-mCherry expressing KIF2C (CINlow) or dnMCAK (CINhigh) BT549 cells (f) or MDAMB231 (g) assessed by live cell imaging, n indicates the number of assessed mitotic cells. h–j) Colony formation assay for isotype control- or 10 µM tocilizumab-treated BT549 (h), MDAMB231 (i), or 4T1 (j) cells expressing KIF2C (CINlow) or dnMCAK (CINhigh). Cells were treated for a week. k) Weights of tumours extracted from immuno-compromised mice with xenografted MDAMB231 cells expressing KIF2C (CINlow) or dnMCAK (CINhigh) treated with either tocilizumab or IgG control. Each dot represents a single tumour. l) Quantification of immunohistochemical staining for STING on KIF2C (CINlow)- or dnMCAK (CINhigh)- expressing MDAMB231 xenografts. To quantify STING levels, cells were classified based on their staining intensity. Negative= no staining, N1= weak staining, N2= moderate staining, N=3 strong staining. m) Quantification of cells with perinuclear STING staining in MDAMB231 xenografts expressing KIF2C (CINlow)- or dnMCAK (CINhigh). n) Representative image for STING staining on an MDAMB231-xenografted tumour. Scale bar equals 50 µm. o) Quantification of immunohistochemical staining for phosphorylated STAT1 (p-STATY701) on KIF2C (CINlow)- or dnMCAK(CINhigh)- expressing MDAMB231 xenografts. To quantify p-STAT1Y701 levels, cells were classified based on their staining intensity. Negative= no staining, N1= weak staining, N2= moderate staining, N=3 strong staining. p) Representative image for p-STAT1Y701 staining on an MDAMB231-xenografted tumour. Scale bar equals 50 µm. q–r) Weights for tumours extracted from immunocompromised athymic (q) or immuno-proficient Balb/c (r) mice with xenografted 4T1 cells expressing KIF2C (CINlow) or dnMCAK (CINhigh), treated with either tocilizumab or IgG. Each dot represents a single tumour. Error bars (b, h–m, o, q–r) represent the s.e.m., n=3 biological replicates (b–e, h–j), n=5 biological replicates (l–m, o), the number of replicates n is indicated in the panel (k, q, r), significance was tested using a two-sided t-test, * P<0.05, ** P<0.01, *** P<0.005, exact P-values are specified in Source Data Extended Data Fig. 8. Cells were treated for 96 hours (b–e).

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Extended Data Fig. 9 Correlations between CIN, aneuploidy, and IL6 and IL6R expression with cGAS expression or interferon activity in TCGA breast cancers.

Expression levels of a gene (Variance Stabilizing Transformation) or gene set (Gene Set Variation Analysis, GSVA) are shown on both axes of each panel. Aneuploidy is measured by the average deviation of DNA copy number changes from the sample mean along the genome. Point colour indicates the hormone receptor status (red if both are negative, blue if either one or both are positive), and shape the HER2 amplification status by IHC. Panels labelled as "sample" show the value obtained from the tumour sample, "cancer" denotes the residual effect with the linear effect of sample impurity removed. a) cGAS expression correlates significantly with both aneuploidy and the CIN70 signature genes in the cancer cells, but not with aneuploidy when taking into account cancer and non-cancer cells (whole sample). This indicates that aneuploid cancer cells have a higher expression of cGAS than euploid cells, and this level is higher the more aneuploid the cancer cells are. b) cGAS expression correlates significantly with IL6 and IL6R expression irrespective of looking at the whole sample or the cancer-specific subset. The correlation is stronger in the whole sample, which indicates that IL6 expression is mainly driven by non-cancer cells in an aneuploidy-driven cancer environment, but the cancer cells themselves also increase their IL6 expression with increased aneuploidy. c) Interferon response genes correlate significantly with cGAS, IL6, and IL6R expression when taking into account the whole sample, but not with IL6 and IL6R expression for the cancer-only population. This indicates that cancer-specific IL6 and IL6R expression are independent of the cancer-intrinsic interferon response, potentially due to oncogenic adaptation to CIN4. d) IL6-STAT3 signalling correlates significantly with cGAS, IL6, or IL6R irrespective of looking at the sample or the cancer-specific population. This indicates that cancer cells themselves increase cGAS, IL6 and IL6R expression with increasing aneuploidy.

Extended Data Fig. 10 Tocilizumab selectively kills cancer cells with induced acute or chronic CIN across various cancer types dependent on IL6 and IL6R expression levels.

a) REACTOME gene set enrichment for genes that become less essential with increasing IL6R mRNA levels in a DepMap analysis. Significance was tested using a student’s t-test. b) Relative expression for IL6 and IL6R for all cell lines used in this study assessed by quantitative RT-qPCR. Expression of IL6 and IL6R were normalised to the lowest IL6 and IL6R expressing cell line, SKMEL28. Samples were measured in technical triplicate. Note that the results are in good agreement with the DepMap-annotated expression data, except that OVCAR3 and NCIH1975 cells express more IL6 in our experiment than reported in DepMap. c) Quantification of mitotic abnormalities in indicated cancer cell lines (lung cancers, skin cancers, and ovarian cancers) expressing KIF2C (CINlow) or dnMCAK (CINhigh). d) Control DMSO-normalised cell viability of DMSO- or reversine-treated cells treated with increasing concentrations of tocilizumab. Top row: lung cancer cell lines, middle rows: ovarian cancer cell lines, and bottom row: skin cancer cell lines. JHOC5, A375, A2780, RMGI, OVCAR3, OVMANA, MEWO, and SKMEl28 were treated with 150 nM reversine. HCC827, H292, SKOV3, and NCI-H1975 with 250 nM reversine. e) Control DMSO-normalised cell viability of A2780 (ovarian cancer), NCI-H1975 (lung cancer), or A375 (skin cancer) treated with tocilizumab concentrations up to 1 mg/ml. f) Stratification of patient survival for aneuploid (left) and euploid (right) lung (LUAD, LUSC; top two panels), ovarian (OV, middle two panels) and skin cancers (melanoma; SKCM, bottom two panels) in the TCGA, matched for the functional cell line validation. Lung and ovarian tissues show a significant decrease in overall survival with active cancer-cell intrinsic IL6 signalling, melanoma does not. No tissue showed a survival effect with cGAS/IL6 for euploid cancers, but it should be noted that the sample number of euploid skin and ovarian cancers is low. g) Relative expression (z-score) of cGAS, IL6 and IL6R across the tissues used for validation in the TCGA. Lung cancers show high expression of all three, ovarian moderate expression of cGAS and IL6. Melanoma shows low expression of cGAS, potentially explaining the lack of cGAS/IL6-driven survival difference in (f). Boxes represent median +/− quartiles, whiskers indicate the 1.5 inter-quartile range. h) Weights for tumours extracted from immuno-compromised mice with xenografted HCC827 lung cancer cells expressing KIF2C (CINlow) or dnMCAK (CINhigh) treated with either tocilizumab or IgG control. Each dot represents a single tumour. i) Relative expression for IL6 and IL6R for primary T-ALL cultures used in this study assessed by quantitative RT PCR. Expression of IL6 and IL6R were normalised to the lowest IL6 and IL6R expressing T-ALL culture, Msh2−/− (eT) cells. Samples were measured in technical triplicate. Error bars in (d–e, h) represent the s.e.m, n=3 biological replicates (d–e), number of replicates n is indicated in the figure (g), significance was tested by a two-sided t-test. * P<0.05, ** P<0.01, *** P<0.005, exact P-values are specified in Source Data Extended Data Fig. 10. Cells were treated for 96 hours (d–e).

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Hong, C., Schubert, M., Tijhuis, A.E. et al. cGAS–STING drives the IL-6-dependent survival of chromosomally instable cancers. Nature 607, 366–373 (2022). https://doi.org/10.1038/s41586-022-04847-2

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