Inhibition of the ALT pathway by interfering with Poly-ADP-Ribose metabolism

ABSTRACT Cancer cells must activate a telomere elongation mechanism and acquire genomic alterations to survive. Many of the most lethal cancers rely on the Alternative Lengthening of Telomeres (ALT) telomere elongation pathway. ALT is strongly associated with recurrent mutations in genes encoding chromatin modifiers such as the ATRX- DAXX chromatin remodeling/histone deposition complex. These disrupt the assembly of telomeric chromatin, provoking replicative stress and double-strand breaks (DSBs) that stimulate specialized homology-directed DNA repair (HDR) mechanisms to repair and extend telomeres. Thus, the ALT pathway represents a valuable target for cancer therapy development. Despite significant advances in understanding these unique ALT-associated HDR (ALT-HDR) mechanisms, the ALT pathway remains unexploited for cancer therapy development. The mono-ADP-ribosylation (MARylation) of chromatin has recently emerged as a key sensor and initiator of the DNA damage response. MARylation predominantly occurs on serine residues and relies on HPF1, which interacts with PARP1. MARylation of specific serines on histones seeds further PARylation that destabilizes nucleosomes proximal to DNA double strand breaks (DSBs) and licenses the recruitment of the first wave of DNA repair complexes. ARH3 is the only known enzyme capable of removing MAR synthesized by the HPF1-PARP1 complex. Despite biochemical evidence supporting its fundamental role in genome maintenance, the identity of additional cellular targets of serine MARylation, and effects of its deregulation, remain obscure. In Aim 1 we will assess the impact of ARH3 and HPF1 disruption on ALT-HDR mechanisms and survival of ALT cancer cells. In Aim 2, we will employ novel approach to track histones as they are being deposited at telomeres and evaluate the impact that this has on transcription of TERRA and the recruitment of major mediators of ALT. We will also employ innovative proteomics to identify telomeric targets of MARylation. The successful completion of these aims will contribute to the understanding of this important chromatin mark during ALT-HDR and its impact on cancer cell survival.