CHD4 Loss Alters Chromatin Accessibility Giving Rise to Sensitivity to DNA Damage Repair Compounds in Acute Myeloid Leukaemia

Background: Acute Myeloid Leukaemia (AML) is a heterogeneous group of blood and bone marrow disorders arising from myeloid precursors. Responses to current therapeutic options remain sub-optimal for many individuals, highlighting the need for novel therapeutic options; alongside stratifying patients by (epi)genetic alterations. Chromatin remodelling complexes (e.g. nucleosome remodelling and deacetylation complex (NuRD)), that control chromatin organization and gene expression, have emerged as key players in cancer. Cancer genome studies highlighted that modifications in NuRD occur in > 20% of cancers. NuRD regulates chromatin accessibility; CHD4, a core subunit, has a multitude of functions including epigenetic regulation, DNA Damage Repair (DDR), replication and cell cycle progression.

Aims: To explore the molecular and epigenetic functions of CHD4 in AML using CRISPR-Cas9 isogenic knockout models (CHD4^-/-), particularly the role of CHD4 on AML development and progression and evaluate if there is targetable therapeutic potential.

Methods: TGCA datasets were assessed to identify impact of CHD4 expression on AML patient prognosis. Isogenic CHD4^-/- models were generated using CRISPR-Cas9 in two AML cell lines (OCI AML 2 and OCI AML 3). Knockout models underwent functional phenotyping studies before inducing DNA damage at clinically relevant doses. Epigenetic mechanisms of CHD4 were assessed using multi-omic approaches including ATAC sequencing, RNA sequencing and proteomic analysis. A DNA Damage/Repair Compound Library (Selleckchem) was screened on CHD4^-/- and CHD4 WT models using CellTox™ Green to identify DNA damage compounds that showed significant sensitivity in CHD^-/- models and validation was assessed using CellTiter® Glo viability assay and co-culture assays. Combination indices (CI) were calculated using CompuSyn software.

Results: Analysis of TCGA AML transcriptomic data (N=173), found that AML patients with intermediate risk cytogenetics and low CHD4 expression had a poorer 5-year survival compared to similar risk group patients with higher CHD4 levels (P=0.032). This indicates that lower CHD4 levels may result in a link between altered cellular transcription and patient survival causing a more aggressive disease phenotype in these patients.

Phenotypical analysis of CHD4^-/- models showed increased 53BP1 (P= 0.026) and PARP indicating DDR defect characterised by unrepaired dsDNA breaks. Defective DDR was shown by reduced proliferation with cell cycle analysis showing inter-S phase defect with accumulation in G1 and reduction in cells entering S and G2/M phases in the CHD4^-/- models. With this profile being further exacerbated under cell exposure to clinically relevant irradiation induced stress.

Chromatin accessibility was assessed via ATAC sequencing and showed enhanced open chromatin at areas impacting immune regulation within CHD4^-/- cells. RNA and proteomic analysis both showed upregulation of genes involved in immune activation (P=6.96e-8) and cellular senescence (P=5.6e-5) pathways suggesting an altered immune profile in CHD4-/- cells with a potential to exploit this therapeutically.

To identify potential therapeutic vulnerabilities in CHD4^-/- cells we performed high throughput screening using a 160 compound DNA damaging library which showed an increased sensitivity of CHD4^-/- cells to compounds targeting DNA/RNA synthesis pathways, including Mitomycin C, this indicated that loss of CHD4 results in hyper-increase in damage. Further investigation with Mitomycin C found that when in combination with Cytarabine yielded enhanced synergistic effects in CHD4^-/- cells (CI 0.001-0.37) when compared to CHD4 WT (CI 0.002-3.1). There was also significantly less viable cells (P=0.016) 24-hours post combination treatment in CHD4^-/- compared to WT (33% vs 56%), indicating that CHD4 loss is furthering the damage caused by improper cell cycle leading to cell death.

Conclusion A cohort of AML patients with intermediate risk cytogenetics along with lower CHD4 levels have poorer 5-year survival. Lack of CHD4 is shown to cause epigenetic restructuring, gene abnormalities and defective DDR. We have identified Mitomycin C as a therapeutic potential in combination with Cytarabine in CHD4-/- models. Further investigation of this in combination and the mechanism of action could lead to improved outcome for this sub-group of patients.

No relevant conflicts of interest to declare.