Abstract Background Unrepaired DNA damage leads to the accumulation of cytosolic DNA and the activation of cGAS-STING-mediated inflammatory response, which modulates the tumor immune microenvironment and immunotherapy response. NPRL2 (NPR2 like, GATOR1 complex subunit) is located on chromosome 3p. Deep deletion of NPRL2 was identified in 10% of ccRCC, and is often mutually exclusive with loss of PBRM1, SETD2, and BAP1. However, it remains unclear whether NPRL2 loss independently influences DNA damage and anti-cancer immunity in ccRCC. Methods Both lentiviral shRNA-mediated knockdown and CRISPR/Cas9-mediated knockout were performed in human and murine RCC cell lines to suppress NPRL2 expression. The immunocompetent Renca-Balb/c murine RCC model was used to evaluate therapy response. The tumor immune microenvironment was analyzed using gene expression-based inference and immunohistochemistry (IHC). Immunofluorescence, western blotting and real-time PCR were used for molecular studies. Results Guided by our reverse phase protein array (RPPA) results, we first found that NPRL2 loss activated the ATM-CHK2 DNA damage response pathway and led to the accumulation of cytosolic DNA. The lysosome is known to play important roles in DNA repair and cytosolic DNA degradation. We further found that NPRL2 loss promoted the phosphorylation of TFEB, the master regulator of lysosome biogenesis, and thus prevented its translocation to the nucleus. Pharmacologic activation of TFEB with ML-SA1 reduced ATM phosphorylation and cytosolic DNA accumulation, and lysosomal inhibition by chloroquine or bafilomycin A1 yielded the opposite results. However, genetic inhibition of autophagy by knocking down ATG5, ATG7, BECN1, or p62 had no apparent impact. The increased cytosolic DNA led us to hypothesize that NPRL2 deficiency would consequently activate the cGAS-STING pathway. Unexpectedly, NPRL2 deficiency suppressed the cGAS-STING-mediated inflammatory response, as evidenced by dramatic decreases in TBK1 and IRF3 phosphorylation, and in downstream gene expression (eg, CCL5, CXCL10 and IFNB1). To investigate the underlying mechanism, we first analyzed the expression of the key molecules that are involved in the cGAS-STING pathway. It turned out that NPRL2 loss reduced the expression of STING at both mRNA and protein levels. By analyzing the KIRC TCGA dataset, we found that low NPRL2 expression was associated with reduced T cell infiltration and immune checkpoint (CTLA4 and PDCD1) expression, reflecting an immunologically cold tumor microenvironment, which may respond poorly to immunotherapy. To test this hypothesis, we knocked down the expression of Nprl2 in Renca cells and found that Nprl2 loss reduced the complete response rate to anti-PD1 therapy in a syngeneic tumor model. However, NPRL2/Nprl2 loss sensitized RCC cells and Renca tumors to the ATM inhibitor KU60019, while the combination of KU60019 and anti-PD1 therapy showed no evidence of synergistic effect. Conclusions These results indicate that NPRL2 loss regulates DNA damage and cytosolic DNA accumulation via lysosomal but not canonical autophagic inhibition, and on the other hand, silences cytosolic DNA response by suppressing STING expression. NPRL2 loss defines a genomically unstable but nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in RCC.
Liu et al. (Wed,) studied this question.