PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

A Di Masi,F Lo-Coco,R Pennisi,D Cilli,S Minucci,S Leone,P Ascenzi,A Antoccia,N I Noguera,I Pallavicini,F Berardinelli,A Talarico,C Nervi

doi:10.1038/cddis.2016.115

Abstract

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Highlights

The DNA damage response (DDR) includes cell cycle arrest and transcriptional and post-translational activation of genes involved in DNA repair and triggering of apoptosis
Double immunofluorescence staining with antiPML and anti-γ-H2AX antibodies allowed the detection of disassembled promyelocytic leukemia (PML)-NBs and high number of γ-H2AX foci in primary blasts isolated at diagnosis from three acute promyelocytic leukemia (APL) patients, harboring the chromosomal translocation t(15;17) resulting in the PML-retinoic acid receptor-α (RARα) fusion product
Disruption of PML-NBs by PML-RARα is a hallmark of APL, a model disease to understand leukemogenic pathways directed by an oncoprotein.[24,25,26,27,28,29,30,31,32]

Summary

Introduction

The DNA damage response (DDR) includes cell cycle arrest and transcriptional and post-translational activation of genes involved in DNA repair and triggering of apoptosis. The sensing phase of the DSB response includes their recognition by the MRE11/RAD50/NBN complex, ATM protein activation, histone H2AX phosphorylation at Ser[139] (γ-H2AX), MDC1, and 53BP1 recruitment. PML-NBs are nuclear organelles implicated in cellular processes relevant to tumor suppression (e.g., PTM, DDR, transcriptional regulation, induction of apoptosis, and senescence).[16,17,18,19,20,21] PML-NBs increase in number and change their subnuclear distribution in response to DNA damage and represent structures where protein complexes are assembled, anchored, and/or post-translationally modified.[11,13,15,16,22]. PML undergoes PTMs, SUMOylation representing one of the main modification necessary for the proper NB biogenesis and regulation of the cellular response to the DNA damage.[23]

Methods

Results

Conclusion