Exploiting Replication Stress as a Novel Therapeutic Intervention

Abstract

Ewing sarcoma is an aggressive pediatric tumor of the bone and soft tissue. The current standard of care is radiation and chemotherapy, and patients generally lack targeted therapies. One of the defining molecular features of this tumor type is the presence of significantly elevated levels of replication stress as compared with both normal cells and many other types of cancers, but the source of this stress is poorly understood. Tumors that harbor elevated levels of replication stress rely on the replication stress and DNA damage response pathways to retain viability. Understanding the source of the replication stress in Ewing sarcoma may reveal novel therapeutic targets. Ewing sarcomagenesis is complex, and in this review, we discuss the current state of our knowledge regarding elevated replication stress and the DNA damage response in Ewing sarcoma, one contributor to the disease process. We will also describe how these pathways are being successfully targeted therapeutically in other tumor types, and discuss possible novel, evidence-based therapeutic interventions in Ewing sarcoma. We hope that this consolidation will spark investigations that uncover new therapeutic targets and lead to the development of better treatment options for patients with Ewing sarcoma. IMPLICATIONS: This review uncovers new therapeutic targets in Ewing sarcoma and highlights replication stress as an exploitable vulnerability across multiple cancers.

Figure 1.. Canonical activation of the ATR-CHK1 signaling pathway.

In its simplest form, the ATR-CHK1 pathway consists of three main steps. The first is the generation of ssDNA gap between the MCM2–7 helicase complex and the DNA polymerase. The generation of ssDNA gap can occur on either leading or lagging strand but most often occurs on the leading strand due to the discontinuous nature of lagging strand synthesis. Then, the ssDNA is recognized and bound by the heterotrimer RPA complex which is the substrate that facilitates the binding of the ATR-interacting protein, ATRIP. ATRIP binding catalyzes the recruitment of the ATR kinase to the site of the stalled fork. ATR binding to chromatin facilitates the activation of its kinase activity. Active ATR phosphorylates several substrates including RPA2, ATR itself and, most importantly, CHK1 kinase. Once phosphorylated by ATR, CHK1 disperses throughout the nucleus, amplifying the signaling cascade. The four main objectives of this pathway are to inhibit the progression of the cell cycle, halt the firing of any late-firing replication origins, promote the initiation of replication from local dormant origins, contributing to various DNA repair pathways, while stabilizing and allowing for the restart of the stalled replication fork.

Figure 2.. ATR-CHK1-WEE1 role in the regulation of replication origin firing.

Even in the absence of replication stress, the enzymes involved in the replication stress response work to limit the level of origin firing by directly and indirectly inhibiting the kinase activity of CDK2-cyclin E and CDC7-DBF4 (DDK). The activation of the replisome requires to main events; the binding of CDC45 and the binding of the GINS complex. Along with the MCM2–7 helicase, the complex formed between CDC45 and GINS is referred to as the CMG helicase complex (CDC45-MCM2–7-GINS helicase complex). The recruitment of CDC45 and GINS requires CDK2- and DDK-mediated phosphorylation of the MCM2–7 helicase. WEE1 mainly works to phosphorylate and deactivate the CDK2-cyclin E complex. This phosphorylation is actively removed by the APC/CDC25A phosphatase complex. Active CHK1 phosphorylates CDC25A, marking it for degradation by the proteasome, indirectly inhibiting the activity of CDK2. ATR directly phosphorylates GINS inhibiting DDK binding, hindering DDK kinase activity and limiting its ability to phosphorylate and active the replisome.

Figure 3.. Histone recycling and replication stress.

Histone recycling takes place during replication and is one of the main mechanisms that allow for epigenetic information that is carried on histones to be inherited by daughter cells upon cell division. A) Parental histones are removed ahead of the progressing replication fork and are immediately deposited into the newly synthesized strands of DNA. These are accompanied by new histones that are initially devoid of any post translational modifications. The methylation pattern of the parental histones is recognized and copied to new histones in a process that still remains somewhat elusive. This allows for the inheritance of epigenetic information to daughter cells and proper recapitulation of gene expression and cellular function after cell division. B) If polymerase progression is impeded by replication blockades the process of histone removal and deposition into newly synthesized DNA is decoupled. This inhibits the histone recycling process. Once the blockade is removed and replication continues, the parental histones that were removed ahead of the fork are no longer in a position to be placed into the new DNA strand. This results in the placement of long stretches of new histones that are devoid of post-translational modifications and loss of the pre-existing epigenetic information. This has the potential to dramatically alter gene expression and cellular function.