RAD51 Degradation Triggered By MALAT1 Silencing Confers Synthetic Lethality to PARPi in AML

Abstract

Listen

Translate article

Introduction Acute myeloid leukemia (AML) critically relies on the activity of RAD51, the central eukaryotic recombinase essential for the homologous recombination (HR) repair of double-strand DNA breaks. PARP inhibitors (PARPi), the first clinically approved drugs designed to exploit the concept of synthetic lethality, selectively target cancer cells with HR deficiencies, particularly those harboring BRCA1/BRCA2 mutations, while sparing normal cell. However, the clinical efficacy of PARPi in AML has been limited, largely due to the infrequency of mutations in DDR genes within these malignancies. Despite this, AML cells exhibit a high accumulation of DNA lesions compared to normal cells, implicating their heavy dependence on DDR pathways, particularly RAD51. Therefore, targeting RAD51 in combination with PARPi in AML cells, even without DDR gene mutations, may exploit synthetic lethality to selectively eradicate leukemic cells while sparing normal hematopoietic cells. Despite the fact that AML cell survival relies on RAD51 overactivation, the regulatory mechanisms governing RAD51 activity in AML remain insufficiently understood. MALAT1, a highly conserved nuclear long non-coding RNA (lncRNA), stands out due to its exceptional evolutionary conservation and extraordinary abundance among lncRNAs. It plays a crucial oncogenic role and is highly expressed across various types of cancer, making it an attractive target for cancer therapy. Methods RNA sequencing was performed on bone marrow samples from seven AML patients and four healthy individuals to identify the genes crucially associated with the progression of AML. Further analyzed MALAT1 expression using two publicly available microarray datasets. Tumorigenicity assays were performed to determine the effects of MALAT1 on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on leukemogenesis or survival of NSG mice xenografted with AML cells (in vivo). Meanwhile, impacts of MALAT1 on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. RNA sequencing to analyze and compare the expression profiles of mRNAs from K562 control and MALAT1 knockout cells. Concurrently, protein mass spectrometry (MS) was conducted to identify differentially expressed proteins between MALAT1 knockout and control cells. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of MALAT1 and RAD51, followed by RNA-pulldown and CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, RAD51/EMI1 interaction and RAD51 ubiquitination in cells in which MALAT1 expression was manipulated. Xenograft mouse models were established by injecting human AML cells into NOG mice via tail vein injection to evaluate the role of MALAT1 and PARPi in the malignant progression of AML in vivo. Results MALAT1 expression positively associates with poor prognosis in AML. Knockout of total MALAT1 resulted in decreased colony formation and proliferation, and increased apoptosis. And cells in G0/G1 phase increased significantly, while the cells in the S phase and G2/M phase decreasedMALAT1 knockout results in decreased. MALAT1 silencing triggers homologous recombination deficiency (HRD) in AML cells. RAD51 protein levels without significant changes in RAD51 mRNA levels, suggesting post-translational regulation may play a key role in its stability. Further investigations demonstrated that MALAT1 directly interacts with RAD51, preventing its ubiquitination and thus stabilizing the protein to promote leukemogenesis. Co-IP experiments suggested that MALAT1 regulates RAD51 ubiquitination by inhibiting the interaction between EMI1 and RAD51. MALAT1 silencing inhibits the progression of AML and triggers PARPi synthetic lethality in AML cells and in vivo. Conclusions MALAT1 modulates RAD51 activity by preventing its degradation, and silencing MALAT1 can induce synthetic lethality in AML cells when combined with PARP inhibitors. This strategy offers a novel therapeutic avenue, though careful management of specificity, toxicity, and resistance is essential for successful clinical application.