Cancer cell-autonomous overactivation of PARP1 compromises immunosurveillance in non-small cell lung cancer

Abstract

BackgroundHigh activity of poly(ADP-ribose) polymerase-1 (PARP1) in non-small cell lung cancer (NSCLC) cells leads to an increase in immunohistochemically detectable PAR, correlating with poor prognosis in patients with NSCLC, as well as reduced tumor infiltration by cytotoxic T lymphocytes (CTLs). Intrigued by this observation, we decided to determine whether PARP1 activity in NSCLC cells may cause an alteration of anticancer immunosurveillance.MethodsContinuous culture of mouse NSCLC cells in the presence of cisplatin led to the generation of cisplatin-resistant PARhigh clones. As compared with their parental controls, such PARhigh cells formed tumors that were less infiltrated by CTLs when they were injected into immunocompetent mice, suggesting a causative link between high PARP1 activity and compromised immunosurveillance. To confirm this cause-and-effect relationship, we used CRISPR/Cas9 technology to knock out PARP1 in two PARhigh NSCLC mouse cell lines (Lewis lung cancer [LLC] and tissue culture number one [TC1]), showing that the removal of PARP1 indeed restored cisplatin-induced cell death responses.ResultsPARP1 knockout (PARP1KO) cells became largely resistant to the PARP inhibitor niraparib, meaning that they exhibited less cell death induction, reduced DNA damage response, attenuated metabolic shifts and no induction of PD-L1 and MHC class-I molecules that may affect their immunogenicity. PARhigh tumors implanted in mice responded to niraparib irrespective of the presence or absence of T lymphocytes, suggesting that cancer cell-autonomous effects of niraparib dominate over its possible immunomodulatory action. While PARhigh NSCLC mouse cell lines proliferated similarly in immunocompetent and T cell-deficient mice, PARP1KO cells were strongly affected by the presence of T cells. PARP1KO LLC tumors grew more quickly in immunodeficient than in immunocompetent mice, and PARP1KO TC1 cells could only form tumors in T cell-deficient mice, not in immunocompetent controls. Importantly, as compared with PARhigh controls, the PARP1KO LLC tumors exhibited signs of T cell activation in the immune infiltrate such as higher inducible costimulator (ICOS) expression and lower PD-1 expression on CTLs.ConclusionsThese results prove at the genetic level that PARP1 activity within malignant cells modulates the tumor microenvironment.

PARP1 inhibition elicits immune responses against non-small cell lung cancer

“…The immune infiltrate of such PARP1 KO LLC cells contained more dendritic cells and more activated, ICOS-expressing CD8 + T cells, but less activated regulatory T cells, coupled to the downmodulation of the exhaustion marker PD-1 on CD4 + and CD8 + T cells. 6 These findings confirm the capacity of active PARP1 to locally suppress the anticancer immune response ( Figure 1c ).…”

“… 3–5 The mRNA levels transcribed from the PARP1 gene are more elevated in non-small cell lung cancers than in adjacent normal tissues, perhaps reflecting an intrinsic resistance of malignant cells against DNA damage. 6 The levels of PAR can be easily assessed by immunohistochemistry. Both in non-small cell lung cancer (NSCLC) and in cervical cancer, high levels of PAR indicate poor prognosis, correlating with a reduced infiltration of the tumors by CD8 + T lymphocytes.…”

“…For this reason, we decided to modulate PARP1 expression/activation in NSCLC cells and to investigate the capacity of PAR high and PAR low NSCLC cells to elicit T cell-mediated immune responses in mice. 6 …”

“…The resulting PAR high NSCLC cell lines maintained their elevated PAR level even after several months of culture in the absence of cisplatin, likely due to an increased level of spontaneous DNA damage. 6 PAR high NSCLC cells were cloned to reduce their heterogeneity and then subjected to the knockout (KO) of PARP1 by CRISPR/Cas9 technology, yielding cells that lacked any signs of PAR accumulation (which demonstrates that PARP1 is the sole PARP isoform generating PAR in these cells) and became sensitive to cisplatin (which demonstrates that PARP1 was indeed responsible for chemotherapy resistance). Of note, PAR high cells exhibited strong responses to the cytostatic and cytotoxic effects of niraparib, a specific PARP1 inhibitor which is clinically approved.…”

“…PAR high LLC cells similarly grew on T cell-depleted and control mice. 6 However, PARP1 KO LLC cells experienced a considerable growth disadvantage when implanted into immunocompetent mice. The immune infiltrate of such PARP1 KO LLC cells contained more dendritic cells and more activated, ICOS-expressing CD8 + T cells, but less activated regulatory T cells, coupled to the downmodulation of the exhaustion marker PD-1 on CD4 + and CD8 + T cells.…”