PARP1 trapping activates cGAS-STING pathway to induce immunogenic cell death in multiple myeloma

Abstract

Background: Alternative Non-Homologous End Joining (Alt-NHEJ) DNA repair is considered a major player in cancer genomic instability. Here, we investigated cGAS-STING pathway as crucial node in the interplay between Alt-NHEJ repair and immune response, in the aim to discover novel therapeutic vulnerability in Multiple Myeloma (MM). METHODS: In silico analyses were performed by querying publicly available MM datasets (GSE66293 and CoMMpass). Anti-proliferative activity was evaluated by CellTiter-Glo, while flow cytometry analysis was used to determine the apoptotic process, cell cycle, phagocytosis, micronuclei detection, Calreticulin and T-cell activation markers. Protein expression was detected by western blot of whole or fractioned protein extracts.

Results: By interrogating public MM datasets, a significant correlation between hyperactivation of cGAS-STING mRNA signature and poor PFS and OS in MM was observed. Indeed, Gene Set Enrichment Analysis (GSEA) showed enrichment of DNA repair, TNFA signaling and oxidative phosphorylation in patients with cGAS-STING activation patients, associated to higher mRNA expression of DNA Ligase 3 (LIG3) and PARP1. On this basis, we evaluated the activity of Alt-NHEJ inhibitor Talazoparib (PARP1-inhibitor) on MM cell lines, focusing on their capability to modulate cGAS-STING pathway. We first detected a significant reduction of cell proliferation and the induction of apoptosis following Talazoparib treatment, which in turn induced DNA damage response and cell cycle blockade, and finally cGAS-STING pathway activation as result of PARP1-trapping into chromatin. Next, by performing co-culture experiments with healthy donor's peripheral blood mononuclear cells (PBMCs), we finally demonstrated the induction of immunogenic cell death, which was abrogated in cGAS-knockout cells, underscoring the pathway's functional relevance.

Conclusion: Taken together, our findings indicate that Alt-NHEJ inhibitors are potential immune-stimulating agents for MM with hyperactivation of cGAS-STING pathway, coherently with our working hypothesis.

Keywords: DNA damage; Hematological malignancies; LIG3; Multiple myeloma; PARP1; cGAS-STING.

Fig. 1

Aberrant cGAS-STING activation is associated with up-regulation of Alt-NHEJ pathway in MM patients. (a) PC dyscrasias’ samples clustering based on cGAS-STING signature expression. Type of samples (N, MM, pPCL, sPCL, HMCL) and MM TC classes (TC1, TC2, TC3, TC4, TC5) and CSG1, CSG2, CSG3, CSG4 groups are depicted in different colors. The color scale bar represents the relative gene expression changes normalized by the standard deviation. (b) Enrichment percentage of CSG signature groups among TC classes (left) and disease stages (right). (c) Enrichment plots of selected significant (nominal p-value < 0.05) gene sets by GSEA analysis on CSG3-4 versus CSG2 groups from CoMMpass dataset. NES values are indicated. (d) Box plots of PARP1 and LIG3 gene expression profiles from CoMMpass dataset. P-value by Kruskal–Wallis test is reported. The dashed black lines indicate significance < 0.0001

Fig. 2

Talazoparib induces DNA damage and cell death in MM cells (a) Indicated MM cells were treated with increasing dose of Talazoparib. CTG assay was performed 5 days from treatment. Results are expressed as percentage of vehicle treated cells. Data are representative of at least three independent experiments. p-values represent the t-tests between each treated point as compared to negative control *, p< 0.05, **p<0.01, ***p<0.01 (b) Primary 138+ cells from 3 MM patients were treated with 2,5 µM of Talazoparib. CTG assay was performed 3 days from treatment. Results are expressed as percentage of vehicle treated cells. Experiments on primary patient-derived cells were performed once per patient due to limited sample availability. (c) Left panel: LP1 and KMS26 cells were treated with increasing dose of Talazoparib. Percentage of Annexin V positive cells 3 days from treatment is shown. Right panel: Representative immunoblot of Cleaved-Caspase 3 and 8 levels was performed 48h after treatment. GAPDH was used as a loading control. (d) LP1 and KMS26 cells were treated with Talazoparib: representative immunoblot of DDR response pathway was performed 48 h after treatment. GAPDH and ACTIN were used as loading control. (e) Left panel: Representative Western blot analysis of nuclear soluble and chromatin bound fractions prepared from LP1 cells treated with Talazoparib (1 µM) for 30’. Histone H3 and LAMININ A/C were used as positive markers for nuclear soluble and chromatin-bound proteins, respectively. Right panel: Quantification (Densitometric units) of Western blot bands, showing relative expression of the target proteins to GAPDH. Data are expressed as means ± SD of three experiments. (f) Cell cycle analysis of KMS26 and LP1 cells after 48h from Talazoparib treatment. Percentage of cell cycle phase are reported. Data are representative of at least three independent experiments. p-values represent the t-tests for G2/M phase of each treated point as compared to negative control. *, p< 0.05, **p<0.01, ***p<0.01

Fig. 3

Talazoparib activates cGAS-STING pathway in MM. (a) Left panel. Micronuclei quantification by flow cytometry 48 h after treatment with increasing concentrations of Talazoparib on LP1 and KMS26 cells. Right panel: Micronuclei analysis by immunofluorescence of LP1 and KMS26 cells, 24 h after treatment with Talazoparib (0.5 µM). Representative immunofluorescence images of LP1 cells showing micronuclei (white arrows). DAPI (blue) was used for nuclear staining. Data are representative of at least three independent experiments. Significance was determined with Student t-test for each treated point as compared to negative control *, P < 0.05, **P < 0.01, ***P < 0.01. (b) LP1 and KMS26 cells were treated with Talazoparib: representative Immunoblot of cGAS-STING pathway was performed 48 h after treatment. GAPDH was used as loading control. (c) Immunofluorescence evaluation p-IRF3 (green) in primary 138⁺ cells from two different patients, after 48 h from Talazoparib (0.5 µM) treatment. DAPI (blue) was used for nuclear staining. Experiments on primary patient-derived cells were performed once per patient due to limited sample availability. Quantification was performed by analyzing at least 50 cells from randomly selected non-overlapping fields. Data are shown as mean ± SD, reflecting intra-sample variability. (d) Left panel: representative western blot analysis of cytosolic, nuclear soluble and chromatin bound fractions prepared from LP1 cells treated with Talazoparib (1 µM) for 72 h. Histone H3 and GAPDH were used as positive markers for chromatin and cytosolic, respectively. Right panel: Quantification (Densitometric units) of Western blot bands, showing relative expression of the target proteins to H3 and GAPDH. Data are expressed as means ± SD of three experiments. (e) LP1 cells were transfected with scramble control or 3 different PARP1-siRNA. Representative immunoblot analysis of cGAS with GAPDH used as a loading control. Analysis was performed 48 h after transfection. (f-g) Percentage of Calreticulin exposure (f) and ATP release (g) in KMS26 and LP1 cells after treatment with Talazoparib for 48 h. Data are representative of at least three independent experiments. P-values represent the t-tests of each treated point as compared to negative control *, p < 0.05, **p< 0.01, ***p  < 0.01

Fig. 4

cGAS mediates anti-MM immunologic response triggered by Talazoparib. (a-b) KMS26 and LP1 MM cells were pre-treated for 24 h with Talazoparib, and then were co-cultured with human DCs and T cells from the same healthy donors for 48 h. CD80 and CD83 expression (MFI) on CD11c positive cells (a) and CD25 and CD69 expression on CD4+ and CD8 ⁺ T cells (b) were reported. Data are representative of at least three independent experiments. Significance was determined with Student t-test for each treated point as compared to negative control *, p< 0.05, **p < 0.01, ***p < 0.01.(c-d) KMS26 and LP1 cells were labelled with CellTrace Far Red, treated with Talazoparib for 24 h , and then co-cultured with human DCs. (c) Phagocytosis analysis reported as percentage of double positive (FAR-RED/CD11c) DCs. Data are representative of at least three independent experiments. Significance was determined with Student t-test for each treated point as compared to negative control *, p < 0.05, **p < 0.01, ***p < 0.01(d) Representative dot plot of phagocytosis assay using flow cytometry. X axis shows FAR-RED + LP1 and KMS26 cells and y axis Cd11 + DCs. (e) Immunofluorescence microscopy analysis from phagocytosis assay. KMS26 and LP1 cells are FAR-RED labeled. (f-g) LP1 WT and cGAS^KO were treated with Talazoparib. (f) Relative cell viability (after 5 days) and (g) % of CALR exposure (after 48 h) are reported as fold change (FC) to NC treated cells. Representative immunoblot analysis of cGAS is shown. Data are representative of at least three independent experiments. Significance was determined with Student t-test for each treated point as compared to negative control *,p < 0.05, **p < 0.01, ***p < 0.01; ns, not significant

Fig. 5

Cartoon describing how Talazoparib could therapeutically interrupt the interaction between cGAS-STING and Alt-NHEJ pathway