Abstract 4451: Stabilization of checkpoint kinase 1 by the lncRNA HOTAIR promotes PARP inhibitor resistance in high-grade serous ovarian cancer

Poly (ADP-ribose) polymerase inhibitors (PARPis) are an important treatment modality for homologous recombination deficient (HRD) cancers, including high grade serous ovarian cancer (HGSOC). PARP mediates DNA single-strand break (SSB). PARPis trap PARP at the site of SSB and prevent DNA SSB repair, which eventually leads to double-strand DNA breaks (DSBs). DSBs are normally repaired through the HR pathway, and PARPis are highly efficient in HRD disease. However, the majority of HGSOC are HR proficient (HRP), and the use of PARPis for patients with HRP tumors represents an unmet therapeutic need. The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is frequently over-expressed in HGSOC and contributes to chemoresistance. We examined the role of HOTAIR in the response to PARPis in HRP and HRD HGSOC cell lines. Knockout or siRNA knockdown of HOTAIR sensitized (p<0.05) HGSOC cell lines to PARPis (olaparib, talazoparib). HOTAIR upregulated the expression of checkpoint kinase 1 (CHK1), a key kinase involved in DNA damage response. Treatment with talazoparib (5μM, 72hrs) increased (p<0.05) expression of CHK1, while HOTAIR depletion with paired CRISPR gRNAs decreased (p<0.05) CHK1 activation by PARPi. In contrast, overexpression of HOTAIR enhanced (p<0.05) talazoparib-induced activation of CHK1. It has been reported that CHK1 inhibition converted PARPi-insensitive cells to PARPi-sensitive cells and, furthermore, that CHK1 interacts with the DNA repair protein RAD51 which was necessary to promote the reversal of stalled replication forks and protect the reversed fork from degradation. Thus, we hypothesized that HOTAIR contributes to PARPi resistance in HGSOC cell lines by regulating CHK1 and CHK1-mediated replication forks stabilization and DNA damage response. We demonstrated that RAD51 expression was reduced (p<0.05) in HOTAIR depletion cells treated with talazoparib, while overexpression of HOTAIR increased (p<0.05) talazoparib-induced activation of RAD51. In addition to a role in replication stress, CHK1 and RAD51 contribute to DNA damage repair through the HR repair pathway. To further examine the role of HOTAIR in DNA damage repair, we examined the effect of PARPi on DNA damage using the comet assay. In HOTAIR overexpressed HGSOC cells, PARPi-induced DNA damage was reduced (p<0.001), based on comet tail moment. As no effect of HOTAIR on CHK1 transcription was observed, we investigated the mechanism of HOTAIR regulating CHK1 by using the cycloheximide chase assay and assessing CHK1 protein stability. HOTAIR depletion increased (p<0.05) degradation of CHK1, and HOTAIR overexpression stabilized CHK1 protein. Collectively, these findings suggest that HOTAIR contributes to CHK1 stabilization, promotes replication fork protection and DNA damage repair in HGSOC and in turn HOTAIR contributes to PARPi resistance, regardless of HR status. Citation Format: Yanchi Zhou, Kenneth P. Nephew. Stabilization of checkpoint kinase 1 by the lncRNA HOTAIR promotes PARP inhibitor resistance in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4451.