DNA replication in cancer cells is often accompanied by stalling and collapse of the replication fork, providing a therapeutic vulnerability if unable to be repaired. Endogenous replication stress occurs when the replication fork stalls, but the MCM helicase continues to unwind downstream causing single stranded DNA. To protect the ssDNA strand from endonucleases, it is coated with RPA, which recruits ATRIP and ATR resulting in the phosphorylation of several mediators of repair including CHK1 and H2AX. If CHK1 signaling is inhibited, cells enter mitosis with damaged DNA (mitotic catastrophe) or replication forks collapse (S-phase catastrophe), both of which result in cell death. There are many causes of replication stalling in cancer cells, including nucleotide depletion, reactive oxygen species or structural hindrance by DNA secondary structure such as G-quadruplex DNA. We, and others, have shown that the MYC/N oncogene causes replication stress in pediatric tumors and relies upon the checkpoint kinases CHK1 and Wee1 to tolerate it.
Subsets of pediatric brain tumors, including high-grade gliomas (HGG), may also be vulnerable to replication checkpoint over-ride. Indeed, AZD-1775 is under investigation as a radiation sensitizer for children with newly diagnosed DIPG (ADVL1217) due to Wee1 overexpression and evidence of single agent Wee1 inhibitor activity and radiation sensitization with external beam radiation in orthotopic models of DIPG. To date, there have been no studies examining ATR/CHK1 inhibition in pediatric high-grade glioma and which factors would predict sensitivity. We hypothesize that ATR, CHK1 and Wee1 inhibitors will have single agent activity and radiation sensitization properties in subsets of pediatric supratentorial PNETs and HGG due to endogenous replication stress from ALT.
What are the goals of this project?
The goals of this project are to test ATR, CHK1 and Wee1 inhibition alone and in combination with radiation therapy in vitro and in vivo in pediatric brain tumor cell lines with and without ALT, to correlate basal replication stress, ATR/CHK1 signaling and ALT in pediatric brain tumor tissues and cell line models of high-grade glioma and sPNET and to perform a loss of function RNAi screen and FDA drug screen in an ALT cell line, with validation in pediatric brain tumor lines with ALT.
What is the impact of this project?
The significance of this work is that, if pediatric HGG and sPNET with ALT are inhibited with ATRi, CHK1i, and this augments radiation sensitivity, it would provide the preclinical rationale to bring forth these inhibitors into a phase I clinical trial. Since ALT is also found in adult lower grade glioma, pancreatic neuroendocrine tumor, neuroblastoma, and subtypes of sarcoma, this work could have a broader application – particularly if downstream signaling is elucidated or biomarkers of response are identified. In addition, the loss of function RNAi screen and FDA screen would potentially identify additional targeted, or synthetic lethal candidates.
The Children's Brain Tumor Network contributed to this project by providing tumor and germline DNA, tissue for cell line generation and cell lines.
Kristina A. Cole, MD, PhD
Kristina Cole, MD, PhD is a pediatric oncologist who has dedicated her career to translational and clinical research, combining her expertise in molecular pathology, cancer genomics, and developmental therapeutics to identify novel treatments for children with cancer. The long-term goal of Dr. Co
Children’s Hospital of Philadelphia
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