Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells

Abstract

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

Keywords: BRCA1 mutant; Cancer; Inflammation; PARP chromatin-bound.

Figure 1.

Veliparib treatment induces upregulation of inflammatory pathways. A. experimental design. Briefly, single cell clones were treated with veliparib for three weeks. Treated cells were then cloned again, grown to confluence, and sequenced. B. GSEA hallmark pathways that are upregulated upon treatment of HCC1937 cells with veliparib. Nominal p < 0.001, false discovery rate (FDR) q < 0.001. C. Quantitative RT-PCR of specific mRNAs.

Figure 2.

HCC1937 cells have increased phosphorylation of TBK1, IRF3 and NFκBp65 upon treatment with veliparib. Quantification of the ratio of phosphorylated p-IRF3/IRF3 (A), p-NFκBp65/NFκBp65 (B) p-TBK1/TBK1 (C), normalized to actin or vinculin. A representative example of a western blot from 3 independent experiments in which cells were harvested after treatment with veliparib for three weeks, as described in methods. Complete scanned gels for western blots are shown in Supplementary Figure S3.

Figure 3.

Long treatment with PARPi increases the presence of cytoplasmic DNA, and cGAS+ micronuclei formation in BRCAm cells. A. Quantification and representative images of cytoplasmic DNA immunofluorescence, dsDNA antibody (dsDNA) and DAPI (DNA). B. γH2A.X foci quantification and representative images. C. Micronuclei quantification and representative images. dsDNA antibody was used to stain micronuclei and the cytosol was stained with CellTrace™ cytosol as was described in the methods section. D. % cGAS+ micronuclei cells. Fold changes were normalized to DMSO controls. Average of 3 independent experiments was considered and 100 cells per experiments were quantified. Bar= 20 μm.

Figure 4.

PARP1 chromatin-bound correlates with PBMC migration. A. Conditioned medium from culturing either BRCA1c or HCC1937 showed significantly increased numbers of PBMC that migrate towards conditioned media isolated from HCC1937 cells treated with veliparib. Data were normalized to DMSO vehicle. B. Quantification of chromatin-bound PARP1 after cellular fractionation and western blotting. Example of a western blot from a fractionation experiment to quantify chromatin-bound PARP1 in chromatin. Fold changes were calculated from the DMSO-treated controls.

Figure 5.

Mutational loads are slightly increased in BRCA1 cells treated with PARPi. A. Graph showing mutation frequencies of different clones of BRCA1c cells and HCC1937 cells (blue) treated with veliparib. Mutational load values were normalized to the DMSO vehicle.

Figure 6.

Veliparib induces tumor immune infiltration in a patient with BRCA1 mutant TNBC. A) Multiplexed quantitative immunofluorescence analysis of baseline TNBC tumor samples (day 0) and after 6 weeks of treatment with veliparib (6 weeks). Fixed tumor specimens were simultaneously stained with a panel containing the markers DAPI (blue), cytokeratin (CK, white), CD4 (red), CD8 (yellow) and CD20 (green). Bar=100 μm. B) The chart indicates the fluorescence level of each immune cell markers before and after treatment. C) Total TCRαβγδ clones and individual clonotypes D) before and after treatment with veliparib. Each TCR chain is indicated with a different color. E) Chromogenic immunohistochemistry analysis of PD-L1 protein using the antibody clone SP142 in the tumor sample before and after treatment with veliparib. Bar=100 μm. F) The chart indicates the percentage of membranous PD-L1 positive tumor and stromal (immune) cells as assessed by a pathologist. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)