Ionizing radiation is a key component to many cancer therapies because it allows doctors to target deeply internal tumors when surgery is not possible. Radiation induces breaks in DNA strands immediately and generates reactive oxygen species that continue to damage DNA for hours. While radiation induces the DNA damage, this, in and of itself does not immediately kill cells, instead the damage initiates one of two major ‘death pathways’. In the first pathway DNA damage activates the well-known tumor suppressor protein, p53 (Trp53), which in turn initiates the apoptosis cascade – an organized proteolytic ‘suicide program’ that does not harm surrounding, undamaged cells. In the second pathway DNA damage is not corrected and cells enter the division cycle, eventually arresting in mitosis due to chromosomal damage, causing mitotic catastrophe – a less well characterized phenomenon that can stimulate either necrosis or a unique subset of the apoptotic pathway. It is essential that scientists and clinicians understand these different mechanisms to improve patient treatment. First, p53 is deleted/inactivated in many tumors treated with ionizing radiation, making them more resistant to radiation therapy. Second, radiation is also toxic to healthy cells, containing functional p53, limiting the doses patients can receive. Thus there is a serious need to make cancer cells more sensitive and healthy cells more resistant to radiation. One of the broad interests for researchers in the Kemp Laboratory here at Fred Hutch (Human Biology Division) is how to better treat p53 null tumors, using cytotoxic therapies including radiation. Their recent study, published in Cell Death and Differentiation, demonstrates that intestinal stem cells require a member of the non-homologous end joining (NHEJ) DNA repair pathway to recover from radiation. Most importantly this pathway was required in both mice expressing and lacking p53.
This study focused on the gene DNA-dependent protein kinase, catalytic subunit (Prkdc). This kinase complex is recruited to DNA damage and phosphorylates many targets in order to activate NHEJ. The Kemp group and others have reported that mice lacking members of the NHEJ pathway are highly sensitive to ionizing radiation, but the reasons for this sensitivity were not immediately clear. Thus the current study compared the response of Trp53 null, Prkdc null, and Trp53/Prkdc null mice to radiation. While many tissues are affected by radiation, the major dose-limiting side effect during radiation therapy is due to cell death within the gastrointestinal tract. Most radiation therapy in human patients is precisely targeted to the tumor site thus dramatically reducing side effects, mice underwent total body irradiation to reduce bias and ensure reproducibility of exposure. Consistent with previous findings, control and Trp53 null mice saw little to no toxicity while all Prkdc null, and Trp53/Prkdc mice died within four days of exposure. When the tissues of these mice were analyzed it was clear that the Trp53/Prkdc compound mutant mice experienced the highest level of toxicity within the gastrointestinal lining. The gut lining is composed of highly organized features called crypts. The base of the crypt contains stem cells that divide infrequently, giving rise to one stem cell and one transit amplifying cell that rapidly divides to generate the differentiated cells that function throughout the gut tissue. Looking closely at the the crypts of irradiated mice revealed two key observations: one, control mice alone had high levels of apoptosis in the more differentiated cell population, and two, the stem cell population was diminished or missing in Prkdc null and even more so inTrp53/Prkdc null mice. This indicated that stem cells require Prkdc and thus NHEJ to survive irradiation, and their death is p53 independent.
Consistently, stem cells within the crypts of Prkdc null and Trp53/Prkdc null mice showed an enrichment of both DNA damage (as indicated by higher levels of γH2A.X) and mitosis (phospho-histone H3) markers. Thus it appears that stem cells rely on NHEJ to repair DNA damage after ionizing radiation, and if this pathway is compromised stem cells arrest in both G1 and mitosis. However, in the absence of p53 all stem cells enter mitosis with DNA damage leading mitotic catastrophe.
The reliance of stem cells on NHEJ was further verified by analyzing the crypts of ATM null and ATM/Trp53 null mice. ATM is an important kinase for activating an alternative DNA repair mechanism, homology directed recombination (HDR). The crypts of ATM null mice exhibited similar behavior to control mice providing more evidence that stem cells primarily respond to DNA damage using the NHEJ rather than HDR pathway. One exciting indication from this work is that inhibiting NHEJ in Trp53 null tumors may improve radiation therapy. The key to this is using targeted radiation and the observation that Trp53/Prkdc null crypts showed a stronger mitotic arrest than Prkdc null crypts. “Radiation can be localized to the tumor to spare normal cells, and this creates a significant therapeutic index: if p53 mutant tumors respond better than p53 wild type normal cells, the dose of radiation or DNA damaging agent may be toxic to tumor cells but spare normal cells” explained Dr. Chris Kemp.
Because p53 mutation/loss is one of, if not the most common alteration in tumors the Kemp Lab is eager to leverage this finding and develop new, targeted therapeutics. "We are using unbiased functional genomic approaches, specifically siRNA screens, to identify additional synthetic lethal genes with p53. These genes may have effective inhibitors that could be repurposed for cancer therapy. We, together with Dr. Eddie Mendez have shown how this works with targeting the WEE1 kinase in head and neck cancer" said Dr. Kemp.
The National Cancer Institute provided funding for this research.
Gurley KE, Ashley AK, Moser RD, Kemp CJ. 2017. Synergy between Prkdc and Trp53 regulates stem cell proliferation and GI-ARS after irradiation. Cell Death Differ. 2017 Jul 7. Epub ahead of print.
Moser R, Xu C, Kao M, Annis J, Lerma LA, Schaupp C, Gurley KE, Jang IS, Margolin A, Biktasova, A, Yarbrough WG, Grandori C, Kemp CJ, and Mendez E. 2014. Functional kinomics identifies candidate therapeutic targets in head and neck cancer. Clin. Cancer Res., 20, 4274-4288, 2014