Targeting PARP1 As an Effective Treatment for Diffuse Large B-Cell Lymphoma with High Level of γH2AX

Introduction

DNA damage repair process has been reported to be the cause of malignant cells resistance to genomic DNA-targeted chemicals. Whether DNA damage repair mediates chemo-resistance in diffuse large B-cell lymphoma (DLBCL) remains elusive. H2AX phosphorylation (γH2AX) is an early event of double-strand DNA repair signaling, which can trigger homologous recombination (HR), classical non-homologous end joining (cNHEJ), or alternative non-homologous end joining (aNHEJ) in a context-dependent manner. The priority choice of PARP1 following H2AX phosphorylation promotes aNHEJ signaling, a well-known oncogenic DNA repair process. PARP inhibitor has increased the efficacy of some neoplasms with HR deficiency. The study addressed the association of γH2AX level with DNA repair signaling and its prognostic value in DLBCL. The role of PARP1 as a therapeutic target was also explored in the study.

Methods

γH2AX protein was examined in new-diagnosed patients with DLBCL (n=90) using immunohistochemistry method. Its prognostic value was evaluated with multivariate analysis. Gene expression profiling was performed on 50 patients and DNA repair signaling was compared based on γH2AX expression. H2AX shRNA was used to downregulate the expression of H2AX and γH2AX through lentivirus in vitro on cell line to observe the effect on doxorubicin-induced apoptosis using flow cytometry. γH2AX and PARP1 were detected using Western blotting and immunoflurescent assay, while DNA repair signaling was detected using PCR array in vitro on cell lines. PARP inhibitor-induced apoptosis was also compared among cell lines using flow cytometry.

Result

γH2AX level suggested a poor prognosis in new-diagnosed patients with diffuse large B-cell lymphoma

γH2AX was detected in 43.34% (39/90) of new-diagnosed DLBCL patients (Figure 1A). Patients clinical features were listed in Table 1. The complete remission (CR) rate was lower in positive patients (31.37% vs 68.62%, p=0.009) after anthrocycline-based chemotherapy. Multivariate analysis showed γH2AX to have an independent inverse association with overall survival after adjusting age, ECOG score, IPI score and Ann Arbor stage in these patients [HR=2.202 (1.161-4.184), p=0.016] (Figure 1B).

DNA repair signaling pathway was enriched in patients with γH2AX expression

Gene expression profiling was performed in 50 new-diagnosed patients with DLBCL. Gene clusters were compared between patients with and without γH2AX expression. The result displayed that DNA repair signaling pathway was enriched in patients with γH2AX expression (NSE=1.901, FDR q-value=0.010) (Figure 1C).

The linkage of γH2AX with DNA repair signaling was confirmed in vitro on cell lines

The expression of H2AX and γH2AX was tested in OCI-ly7, OCI-ly3 and OCI-ly8 cell lines. The highest level of H2AX and γH2AX was observed in OCI-ly7 cells, followed by OCI-ly8 and OCI-ly3 cells (Figure 2A). DNA repair genes were proven to be highly expressed in OCI-ly7 cell line using DNA repair signaling PCR array compared with the other two cell lines (Figure 2B).

Downregulating γH2AX expression increased doxorubicin-induced apoptosis in vitro

The reduction of γH2AX level was demonstrated in H2AX shRNA-transduced OCI-ly7 cell line (Figure 2C). The proportion of doxorubicin-induced (50ng/ml) apoptosis was increased in these cells compared with control (5.87±2.37% vs 39.18±3.78%, p<0.05). (Figure 2D)

PARP1 level presented a positive correlation with γH2AX expression and PARP inhibitor-induced apoptosis in vitro

The level of PARP1 expression was proportional to γH2AX expression in OCI-ly7, OCI-ly3 and OCI-ly8 cell lines (Figure 3A). The result indicates an abnormal aNHEJ oncogenic DNA repair to be superior in DLBCL cells with high γH2AX expression. FEN, another key component of aNHEJ signaling was also overexpressed in cell line and patients with high expression of γH2AX. PARP inhibitor (25μM) induced the highest proportion of apoptosis in OCI-ly7 cell line, compared with OCI-ly8 and OCI-ly3 cell lines (42.90±5.29% vs 29.52±3.95% vs 9.41±2.35%, p<0.05) (Figure 3B).

Conclusion

γH2AX as a biomarker for abnormally activated DNA repair signaling is associated with poor outcome in the setting of conventional chemotherapy in DLBCL. PARP1-mediated aNHEJ signaling performs critical functions in DLBCL with γH2AX expression. Here, we demonstrated that PARP inhibitor may be efficacious in these patients.

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