Synergistic Leukaemia Cell Death Induced with PARP and TiP60 Inhibitors Is a Prospective Therapeutic Strategy for AML

Background: We previously identified pharmacological inhibition of lysine demethylase 4A (KDM4A) sensitises AML cells to inhibition of poly ADP ribose polymerase (PARP), one of the major DNA damage response (DDR) mediators. We hypothesise that synergy between inhibition of PARP and histone acetyltransferase (HAT) family member, Tip60 that is involved in DSB repair, may drive effective AML leukemic cell death sparing normal cells. We aim to unravel molecular mechanisms of action of the targeted inhibitors and downstream targets of Tip60 and PARP in AML.

Methods: Phenotypic changes, such as in cell viability, apoptosis, cell cycle arrest by flow cytometry, colony forming capacity, and DNA damage protein markers by western blotting, in AML cells following drug treatments were determined by standard protocols. Enrichment of biological pathways in which genes with transcriptional changes in AML cells upon the drug treatment were discovered using gene set enrichment analysis (GSEA) of RNA-seq dataset generated from treated cell lines. Downstream target genes of Tip60 and PARP together in common were identified via cross-validation using least absolute shrinkage and selection operator (LASSO) linear regression in clinical datasets.

Results: Loss of PARP and Tip60 enzymatic activities was driven by pharmacological inhibition of PARP and Tip60 individually and in combination in AML cell lines. As a result, a significant synergistic effect of the combination treatment (PARPi plus Tip60i) was confirmed in THP-1 and MOLM-13 cells with combination indices (CI) < 1, which was further validated via the Bliss independent model and isobolograms. Simultaneous combination of PARPi together with Tip60i resulted in significant phenotypic changes with increased early and late apoptotic cell populations, cell cycle arrest at S phase, decreased colony forming ability, and accumulation of DNA lesion markers, γH2A.X and cleaved PARP proteins, which contributed to inhibition of cell growth and ultimately significant increase (p<0.0001) in AML leukemic cell death.

GSEA revealed that combination treatment of PARPi and Tip60i induced significant enrichment of genes (adjusted p-value<0.05) involved in DDR sub-pathways, mismatch repair (MMR; NES=-1.54; FDR=0.088) and interstrand crosslink repair (ICLR; NES=-1.25; FDR=0.22), in parallel, suggesting an inhibitory role of PARPi and Tip60i in impairing DDR in AML cells. Furthermore, global GSEA further demonstrated that synergistic effect of PARPi plus Tip60i induced significant upregulation of biological pathways of hypoxia, unfolded protein response, p53 pathway, and apoptosis; and significant downregulation of oxidative phosphorylation, glycolysis, and Myc pathway.

A minimum subset of 13 genes mediated by inhibition of Tip60 and PARP ('TiP13') in AML cells was constructed in relation to patient survival. Kaplan-Meier survival analysis showed that the gene signature score of each patient in four patient cohorts, calculated as the sum of the weighted gene expression for each gene in TiP13, was highly correlated with patient survival in four test clinical datasets (GSE12417, FDR=0.00091; GSE37642, FDR=0.042; GSE6891, FDR=0.044; Beat AML, FDR<0.0001).

Discussion: Failure to repair DNA damage at transcriptional level by blocking of MMR and ICLR pathways led to constant accumulation of DNA lesions and thereby effective cell killing in AML cells treated with PARPi and Tip60i in combination. This study further demonstrates that transcriptional outcomes driven by synergising PARPi and Tip60i activities contribute to dysregulation of oxidative phosphorylation for oxidation of nutrients and production of ATP in glycolysis in AML cells, leading to genomic instability, accumulated global DNA damage, and increased response to stress or unfolded protein. Consequently, the accumulated DNA lesions alongside impaired capacity of AML cells to repair their damaged DNA, thereby stimulated apoptosis, facilitated with downregulation of an oncogenic Myc pathway and upregulation of tumour suppressor p53 pathway. In addition, this study has uncovered a novel TiP13 gene signature (downstream targets of PARP and Tip60) with prognostic significance in AML that successfully stratifies outcomes of AML patients.

Copland:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees.