DNA methylating drug temozolomide sensitizes ARID1A-mutated tumors to PARP inhibitors (010)

Abstract

<b>Objectives:</b> Somatic mutations of ARID1A are present in greater than 50% of ovarian clear cell carcinoma (OCCC). ARID1A and BRG1 are key subunits that form the SWI/SNF complex to regulate chromatin configuration and other key cellular functions. Somatic mutation of TP53 or BRG1 is rare in OCCC and when either mutation occurs, it is often mutually exclusive with ARID1A mutations, suggesting an overlapping function of these tumor suppressor genes. ARID1A-SWI/SNF complex was involved in DNA damage repair. Compromised DNA repairs are identified in ARID1A-deficient tumors, rendering ARID1A-deficient tumors hypersensitive to PARP inhibitors when primed with low-dose ionizing radiation (IR). The methylating chemotherapeutic drug, Temozolomide (TMZ), induces DNA lesions like IR. Thus, we propose to re-purpose this drug for gynecological cancers. <b>Methods:</b> As a proof-of-principle study, we employed four pairs of isogenic human cell lines, hEM3, MCF10a, HCT116, and OVCA429, engineered with/without ARID1A knockout. HCT116 and OVCA429 have DNA mismatch repair (MMR) deficiency while hEM3 and MCF10a do not. Different concentrations of the PARP inhibitor, Olaparib (Olap), and TMZ were co-applied <i>in vitro</i> to determine cytotoxic synergism. OCCC xenografts were established in athymic <i>nu/ nu</i> mice and treated with TMZ, Olap, TMZ+Olap, or vehicle control. Efficacy was measured by changes in tumor volume. Biomarker and pathological analyses were performed on tumor samples.Fig. 1 <b>Results:</b> MMR-proficient ARID1A-/- cells were more sensitive to TMZ+Olap treatment compared to the isogenic MMR-proficient ARID1A+/+ cells. Additionally, ARID1A-/- cells could not effectively repair DNA lesions induced by the DNA alkylating drug, TMZ, because of a compromised base excision repair. DNA single-strand breaks in ARID1A-deficienct cells were associated with replication fork instability, manifested by fork collapse and replication stress. Moreover, the TMZ+Olap combination treatment delayed the <i>in vivo</i> growth of OCCC xenograft tumors with ARID1A-inactivating mutations. The treatment induced DNA breaks, replication stress, and apoptosis on the xenografted tumors. The markers of replication stress including gH2Ax and pRPA and a marker of apoptosis, cleaved caspase 3, were significantly elevated. Proteomic analysis of ARID1A knockout cells using an anti-BRG1 antibody to isolate the SWI/SNF complex indicated an upregulation of DNA damage repair proteins and RNA helicases, suggesting dysregulation of these processes in ARID1A-deficient cells. <b>Conclusions:</b> Our findings suggest a synergistic relationship between TMZ and Olap in treating ARID1A-deficient tumors. The data demonstrated accumulated DNA lesions and breaks induced by TMZ that cannot be effectively repaired by ARID1A-deficient tumor cells because of hampered base excision repair-related machinery. Thus, ARID1A-deficient tumor cells are highly reliant on PARP-dependent repair. Our data provide pre-clinical supports for a DNA break-primed and PARP inhibitor-based treatment strategy for OCCC.