P476: LOSS OF CHD4 IN ACUTE MYELOID LEUKAEMIA GIVES RISE TO INCREASED DNA DAMAGE REPAIR DEFECTS AND ALTERED CELL CYCLE PROGRESSION

Abstract

Background: Acute Myeloid Leukaemias (AMLs) are an array of blood cell disorders arising from alterations within myeloid precursors. Cancer genome studies highlighted alterations within the nucleosome remodelling and deacetylation (NuRD) complex occur in over 20% of all cancers. The NuRD complex controls nucleosome assembly and chromatin accessibility; CHD4 is a core subunit of the NuRD which is involved in a multitude of functions including epigenetic regulation, cell cycle progression and DNA replication and repair. CHD4 is linked to PARP dependent DNA damage response (DDR) pathway by rapid but transient accumulation at sites of damage, preventing transcription which enables homologous recombination (HR). Aims: We aim to explore the function of CHD4-NuRD in AML using a CHD4 knockout (CHD4-/-) isogenic cell line models created using CRISPR/Cas9 to assess the mechanism of function of CHD4 within AML disease progression and evaluate if there is targetable therapeutic potential. Methods: CRISPR-Cas9 was used to generate isogenic CHD4-/- models in two representative AML cell lines. CHD4-/- models underwent functional studies; proliferation, cell cycle and protein expression assays were used to phenotype models before inducing DNA damage at a clinically relevant dose and reassessing cell response. A high throughput drug screen was performed to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using single compound treatments and co-culture assays. Results: Analysis of publicly available TCGA AML transcriptomic data set (N=161), found that AML patients (N=82) who express below median CHD4 levels have a poorer prognostic outcome suggesting a link between stalled transcription enabling HR-based DDR and patient survival (P=0.047). Analysis of the intermediate cytogenetic risk group showed patients with a low CHD4 expression had a worse outcome; which was comparable to the adverse risk group patients (P=0.032), indicating that loss of CHD4 may contribute to a more aggressive phenotype AML in this group. Phenotype analysis of CHD4-/- models showed DNA damage markers 53BP1 and PARP expression increases significantly, highlighting the role of CHD4 within the DDR. This increased DNA damage response was accompanied by reduced cellular proliferation in CHD4-/- cells. Accordingly, cell cycle analysis showed a decrease in cells entering the G2/M phase alongside an increase in cells in G1 and S phase suggesting a potential cell cycle checkpoint deficiency causing stunted proliferation in CHD4-/- cells. Of note, we did not observe a significant compensatory increase in CHD3-NuRD components expression in the absence of CHD4 suggesting there is no compensation for the loss of CHD4. We measured behaviour of CHD4-/- cells to stress-inducing conditions; in response to irradiation and chemotherapy drugs commonly used in AML. CHD4-/- cells display much stronger reduction in cellular proliferation post-2Gy irradiation compared to control while cell cycle analysis showed further cellular arrest in G2/M. Furthermore, a drug screen (160 DNA damaging compounds) showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Tubercidin and Cytarabine, indicating that loss of CHD4 results in hyper-increase in damage. Summary/Conclusion: Our data has identified a role of CHD4 in AML progression mediated through loss of cell cycle progression control and an elevated response to DNA damage. Therefore, targeting cells with lower CHD4 expression with compounds targeting DNA/RNA synthesis may have an impact on patient outcome.