Studies On Antitumor And Resistance Mechanisms Of PARP Inhibitors

Poly?ADP-ribose?polymerase inhibitors?PARPis?are novel small molecule targeted drugs based on synthetic lethal interaction.At present,four PARPis have been approved for cancer therapy,and have achieved great successes in the treatment of ovarian cancer,breast cancer,pancreatic cancer,etc.PARPis have occupied an important position in the field of antitumor therapies.However,the antitumor mechanism of PARPis has not been fully elucidated.For instance,PARP1-DNA trapping is controversial.Moreover,tumors will inevitably develop resistance to PARPis,leading to treatment failure.However,the characteristics and mechanisms of PARPi resistance remain to be explored more extensively.This study systematically studied the antitumor mechanism of PARPis,elucidated the key factors determining the cytotoxicity of PARPis,and developed a more effective high-throughput molecular screening model.Moreover,the mechanisms of PARPis resistance in BRCA2 deficient pancreatic cancer Capan-1 cells were studied.A BRCA2 intron new mutation and apoptosis resistance,which led to PARPi resistance,were uncovered.In terms of the antitumor mechanism of PARPis,we clarified the relationships between cytotoxicity,PARP1-DNA trapping,PARP1 enzymatic activity and its inhibition.The PARP1-DNA trapping intensity of PARPis was shown not to be strongly correlated with their cytotoxicity.Three different strategies,including same concentration of different PARPis,different concentrations of the same PARPi,and the corresponding concentration at the respective IC₅₀?reflecting cytotoxicity?of different PARPis were used to treat different cancer cells and subsequently detected PARP1-DNA trapping.Correlation analyses showed that no significant correlation occurred between cytotoxicity and PARP1-DNA trapping.In contrast,the cytotoxicity of PARPis is correlated with cellular PARP1 enzymatic activity.We constructed PARP1 knockout monoclones of Ewing's sarcoma,and then stably complimented the wild-type PARP1?WT?,the mutated PARP1?E988K?,and the PARP1 mutants with different enzyme activity.Using parental cells,PARP1 knockout cells,and 12 PARP1mutant cells,we investigated the relationships between cellular PARP1 enzymatic activity,inhibition of PAR formation,PARP1-DNA trapping,and PARPis's cytotoxicity.A further analysis showed that PARPi's cytotoxicity was correlated with cellular PARP1 enzymatic activity most tightly?r=-0.70;p=0.0052?,with its PARP1-DNA trapping moderately?r=-0.58;p=0.0284?and with its inhibition of PAR formation least?r=-0.13;p=0.72?.In addition,PARP1-DNA trapping was also highly correlated with cellular PARP1 enzymatic activity?r=0.82;p=0.0003?and was correlated with weaker inhibition of PAR formation?r=0.35;p=0.33?.Inhibition of PARP1 enzymatic activity by different PARPis measured by the classical histone-based substrate ELISA assay did not match its cytotoxicity?r=0.3898;p=0.4245?.We constructed two fluorescence anisotropy?FA?molecular assays and tested the EC₅₀ of different PARPis in inhibiting the dissociation of WT-PARP1 from DNA?DSB-FA assays?,and their respective average IC₅₀ values in killing 17 PARPi-sensitive cell lines reported.Correlation analyses showed a highly significant correlation?r=0.9984;p<0.0001?occurring between the EC₅₀ values of 5PARPis?except niraparib?in inhibiting the dissociation of WT-PARP1 from DNA and their cytotoxic IC₅₀ values.Detailed analyses indicate that increased PARP1-DNA binding due to autoPARylation inhibition of PARP1 on DNA rather than PARP1-DNA trapping is correlated with PARPis's cytotoxicity.Finally,we compared the two FA assays with the existing PARPis molecular evaluation assays and found that the DSB-FA assay is a new,efficient,rapid,stable,low-cost,non-radioactive molecular screening assay for PARPis,which can more closely predicts PARPi's cytotoxicity.In terms of PARPi resistance,we investigated the characteristics and mechanisms by establishing Capan-1 sublines resistant to PARPis Olaparib?OP?,Simmiparib?SP?and Talazoparib?TP?.Their corresponding sublines were denoted by Capan-1/OP,Capan-1/SP and Capan-1/TP,respectively.These resistant sublines lacked obvious or uniform changes in their cell morphology,proliferation rate and migration ability.Three PARPi-resistant variants displayed different degrees of cross-resistance to all the tested PARPis.In these resistant variants,PARP family members and multidrug resistance protein P-gp did not change significantly;moreover,their BRCA2 mutation site c.6174delT showed no back mutation or missing either.Whole-genome sequencing revealed a new heterozygous mutation in the BRCA2 intron 11,which resulted in the formation of a new truncated BRCA2 splice isoforms with the C-terminus.Functional studies revealed that only a fraction?32.22–49.03%?of PARPi sensitivity could be rescued by depletion of the new C-BRCA2 splice isoforms.Our data showed that higher levels of DNA damage accumulated in the PARPi-resistant variants,which did unexpectedly not affect cell survival and senescence,suggesting that these variants evolved capabilities to tolerate the accumulated DNA damage.Equivalent concentrations of PARPis caused DNA damage accumulation and cell cycle arrest in resistant variants equal to or even higher than those in their parental cells.In contrast,equivalent concantrations of PARPis induced apoptosis in the resistant variants?including phosphatidylserine eversion,caspase 3/7/8/9 activation,and mitochondrial membrane potential loss?significantly lower than that in their parental cells.Further studies revealed that the mRNA and protein levels of the anti-apoptotic proteins COX-2 and BIRC3 in PARPi-resistant variants were significantly higher than those in the parental cells.Depletion of COX-2or BIRC3 significantly reduced apoptosis resistance in the resistant sublines and reversed PARPi resistance by up to 70.31–72.33%,suggesting that overexpression of COX-2 and BIRC3 is one of the mechanisms of PARPis resistance.The combination of the BIRC3 inhibitor LCL161 and PARPis significantly enhanced the sensitivity of the resistant variants to PARPis,providing new clues for the treatment of PARPi-resistant tumors.Additionally,our data further showed that both PARPi treatment and PARP1 knockout markedly increased the mRNA levels of COX-2 and BIRC3,suggesting that PARP1 is a novel negative transcriptional regulator of these genes.Taken together,this study demonstrates that the key factor in determing the antitumor activity of PARPis is cellular PARP1 enzymatic activity.Moreover,increased PARP1-DNA binding due to autoPARylation inhibition of PARP1 on DNA rather than PARP1-DNA trapping is correlated with PARPi's cytotoxicity.Based on this conclusion,we established a new PARPi screening assay that more closely predicts cytotoxicity.This study also uncovers new resistance mechanisms of PARPis:resistant cells evolved a novel mutation in intron 11 of BRCA2 and overexpressed anti-apoptotic proteins COX-2 and BIRC3.Our findings give new insights into the antitumor and resistance mechanisms of PARPis,which might promote the development and application of new PARPis and help to establish new strategies to overcome PARPi resistance.