3.1 Elevated HR and PD-L1 expression is responsible for PARPi resistance.
According to our test in Fig3a by GraphPad Prism software, the IC50 values of olaparib in Cal27 and Scc25 cells were 36 µM and 98 µM at 48h, respectively, indicating that the sensitivity of Cal27 cells to drug was relatively stronger than Scc25 cells. Then the CCK8 assays verified that olaparib alone had no significance effect on OSCC cells’ survival. (Figure_1A)
To search for potential resistance mechanism, treating cells with olaparib at various concentrations and evaluated levels of RAD51, BRCA1 and TOPBP1, observing that the upregulation of these genes obviously compromised the olaparib-mediated killing effects. In Cal27 cells, 5 µM olaparib treatment resulted in increased RAD51, BRCA1 and TOPBP1 (Figure_1B, 1C), causing drug resistance to olaparib as shown in Figure_1A. Similar results were observed in response to 10 µM, although to a lower degree compared with 5 µM olaparib, which can help explain why the treatment effect of 10 µM was better than 5 µM olaparib in CCK8 assay. In Scc25 cells, olaparib at either 10 µM or 20 µM upregulated the expression of RAD51, BRCA1 and TOPBP1 obviously, so the treatment effect of olaparib was worse than that in Cal27 cells. (Figure_1B)
To further explore the mechanism of olaparib resistance in OSCC, we examined the PD-L1 expression and it was consistent with HR-related proteins. (Figure_1B) We also examined the mRNA expressions of RAD51 and CD274 (PD-L1), which had the same trend of proteins in both cell lines. (**P < 0.01, *P < 0.05, Figure_1C)
Moreover, BRD4 expression was also upregulated by olaparib, which indicated the opportunity that we might use BET inhibitor to augment treatment effect of olaparib in OSCC. (Figure_1B)
Together these results indicated that the PARP inhibitor olaparib induced enriched HR-mediated DNA damage response as well as high PD-L1 expression, counteracting drug’s killing effects and resulting in acquired resistance.
3.2 BET inhibition impairs HR-mediated DNA damage repair and PD-L1-medicated immune escape
To evaluate the HR efficiency and PD-L1 level in OSCC cells after treating by BETi, a dual experiments including Immunofluorescence and Western-blot were performed. The results showed that BRD4 pharmacological inhibition using JQ1 induced a dose-dependent reduction in RAD51, BRCA1 and TOPBP1 levels and was able to hinder homologous recombination-mediated DNA damage repair in OSCC cell lines, generating massive cell death and better treatment results. Moreover, the expression of PD-L1 was also prohibited by using JQ1 in a dose-dependent manner, which had similar reaction with HR-relevant factors, verifying that JQ1 could trigger immune inhibition in OSCC cells due to absence of BRD4. (Figure_2)
3.3 BET inhibitor and PARP inhibitor synergistically suppress cell proliferation
We next examined the effects of treating cells with olaparib, the BET inhibitor JQ1, or combining both inhibitors. The IC50 values of olaparib were calculated and they were robustly reduced after combination treatment (**P < 0.01) (Figure_3A), indicating that OSCC cells became more sensitive to olaparib. CI calculations confirmed that the combination JQ1 and olaparib treatment exhibited a synergistic effect (Table 1).
The JQ1 and olaparib treatment simultaneously also caused colony formation inhibition compared with other groups. (Figure_3B) The action of the combined treatment on proliferation was tested using CCK8 assays. The results showed that combination of two drugs cause a remarkable decline in cells’ proliferation, comparing with individual inhibitors (**P < 0.01, Figure_3C).
These results demonstrated that both olaparib and JQ1 at low concentrations had moderate impact on decreasing cell proliferation and viability of Cal27 and Scc25 cells when they were used alone, whereas simultaneously treated with JQ1 greatly triggered cell death and sensitized the OSCC cells to the drug.
3.4 BET inhibitor and PARP inhibitor synergistically induce apoptosis, which correlates with higher p53 levels
We next examined whether the combination treatment affected the apoptosis of OSCC cells. Western blot analysis uncovered that the cleaved caspase-3 which act as an important apoptosis marker was activated at a dramatically higher level in Cal27 and Scc25 cells after treating with JQ1 and olaparib. (**P < 0.01, Figure_4A, 4B) Our research also evaluated cell apoptosis by Annexin V-PE staining followed by flow cytometry in OSCC cells treated with variable inhibitors for 2 days. Apoptotic cells accounted for higher proportions in response to the combination treatment than treatment by either inhibitor alone. (**P < 0.01, Figure_4C, 4D)
P53 signaling is a key pathway that induces apoptosis. We thus next examined whether apoptosis was mediated by the p53 pathway. The combination treatment led to prominently increased p53 and phosphorylated p53 expressions compared with single treatments. (*P < 0.05, **P < 0.01, Figure_4A, 4B)
Together our findings suggest that the combination of BET inhibitor and PARP inhibitor triggered a strong synergism in OSCC cells represented by a marked increase in cell apoptosis, which correlated with activation of the p53 pathway.
3.5 BET inhibitor and PARP inhibitor co-treatment results in cell cycle arrest
Then we focused on the effects of the combination treatment on cell cycle in OSCC cells. The results are displayed in Figure. 5A and quantitative analysis is shown in Figure. 5B. JQ1 and olaparib alone resulted in G1 or G2/M phase cell cycle arrest, respectively, while the combination treatment caused G1 and G2/M phase arrest as well as a significant decline in S phase cells.
Based on our data above, the simultaneous impairment of both HR and PD-L1 pathways is able to induce substantial cell cycle arrest in OSCC cells as shown by the co-treatment with JQ1 and ola.
3.6 Combined JQ1/olaparib inhibition is effective in vivo
Cal27 and Scc25 cells were injected into Six-week-old female mice and after the tumor volume reached approximately 150–250 mm3 after one week, mice were randomized into groups and treated with different inhibitors for 3 times. By the end of the 20 day treatment period, JQ1 and olaparib resulted in modest inhibition of tumor growth for both OSCC cell lines, and this was accompanied by minimal change in PD-L1 and RAD51 levels showed by immunohistochemical staining. The dual treatment was superior to any of the single treatments and resulted in significant tumor shrinkage, which also led to a marked reduction in the expression of RAD51 and PD-L1. (**P < 0.01, Figure_6, 7) The differences were unlikely to be a result of generalized toxicity of drug treatment, since mouse weights were stable over the treatment period for all treatment groups.
The results demonstrated that combined treatment exhibits better antitumor effects in OSCC in vivo by inhibiting HR and PD-L1 pathway, compared with JQ1 or olaparib treatment alone.
3.7 BET inhibitor and PARP inhibitor combination suppresses HR mediated by ATR/CHK1 pathway
To explore the potential mechanism of the enhanced efficacy with combined treatment in OSCC, we examined several key DNA damage response factors involved in HR. Western blot showed that these HR-related molecules were highly downregulated by the combination treatment compared with olaparib alone, although the expressions were slightly higher than in response to JQ1 alone. (Figure_8A) To better quantitate the cumulative expression of RAD51, we performed Flow cytometry and Immunofluorescence assays in Cal27 and Scc25 cells, respectively. Under fluorescence microscope and FACS flow cytometer, OSCC cells displayed the same level of the factor as seen in the Western blot. (Figure_8B, 8C) We also observed decreased RAD51 mRNA level in Cal27 and Scc25 cells treated with the combination therapy compared with olaparib monotherapy (**P < 0.01, Figure_8D), which verified that JQ1 combined with olaparib can act by suppressing HR.
ATR/CHK1 plays a critical role in the regulation of replication and is essential in HR, and thus we subsequently examined the ATR/CHK1 pathway. Consistent with our above results, the ATR/CHK1 pathway was increased in response to olaparib compared with controls, while the combination treatment reversely blocked this pathway compared with olaparib alone (**P < 0.01, Figure_8A, 8D), further indicating that JQ1 maximized olaparib’s treatment effect through impairing HR mediated by ATR/CHK1 pathway inhibition.
These results indicate that the added BET inhibitor JQ1 disrupted HR via depleting ATR/CHK1 expressions, thus mitigating olaporib resistance and sensitizing the OSCC cell to the PARP inhibitor.
3.8 BET inhibitor and PARP inhibitor combination attenuated expression levels of BRD4 and PD-L1
We continued to explore the mechanism behind the combinatorial synergism of JQ1 and olaparib in OSCC cells. The use of JQ1 and olaparib synchronously reduced BRD4 expression to a greater extent compared with JQ1 monotherapy in Cal27 and Scc25 cells, which demonstrated that the sensitivity of OSCC to the BET inhibitor was also enhanced by the combination treatment. (*P < 0.05, **P < 0.01) (Figure_9A, 9D)
We next determined whether BRD4 correlates with PD-L1 expression in OSCC by examining a panel of OSCC cell lines. We observed a trend towards a positive relationship between BRD4 and PD-L1 expression. (Figure_9A) We then examined whether BRD4 and CD274 expression was also positively correlated in OSCC cells. Indeed, there was a significant positive mRNA correlation between BRD4 and CD274 in Cal27 and Scc25 cells treated with JQ1, olaparib or the combination. (Figure_9D) Notably, decreasing PD-L1 expression may be a strategy to optimize the antitumor effects of PARP inhibitors by adding BET inhibition. (Figure_9B, 9C)
Our results showed that the PARP inhibitor upregulated PD-L1 and BRD4 in OSCC cells, while BET blockade mitigated PD-L1-mediated immunosuppression activity, resulting in better treatment effect in OSCC cells. These results verified that the sensitivity of OSCC to the BET inhibitor was enhanced by the combination treatment with the PARP inhibitor.