Katherine D. Castle

Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy

Tumor cells, rather than endothelial cells, are critical targets that regulate primary sarcoma eradication by radiation therapy. Not all cells are eradicated equally Personalized cancer therapies dominate the news. But radiation therapy continues to be an essential part of the treatment regimens of nearly half of all cancer patients—sometimes achieving complete tumor regression through the safe delivery of high doses of radiation. Previous research with transplanted tumor models in mice has suggest that radiation targets, not only the tumor cells themselves, but also components of the surrounding milieu, which comprises blood vessels and various cell types that influence tumor growth. Now Moding et al. challenge the earlier findings in studies conducted with primary sarcomas in mice that carried, in either tumor or endothelial cells, genetic mutations that modulate radiation sensitivity. The authors found that it was the tumor, rather than endothelial, cells that mediate primary sarcoma shrinkage by radiation therapy and that selective small-molecule inhibition of a DNA-damage response enzyme can enhance radiosensitization of some tumors. Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for several kinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remains controversial. Using dual recombinase technology, we generated primary sarcomas in mice with targeted genetic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death in tumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelial cell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication. In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These results demonstrate that tumor cells, rather than endothelial cells, are critical targets that regulate sarcoma eradication by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitized primary sarcomas more than the heart. These results suggest that inhibiting ATM kinase during radiation therapy is a viable strategy for radiosensitization of some tumors.

Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma

Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.

Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in Mice

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3′-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit+ Lin– Sca1+ (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Dual-Energy CT Imaging of Tumor Liposome Delivery After Gold Nanoparticle-Augmented Radiation Therapy

Gold nanoparticles (AuNPs) are emerging as promising agents for both cancer therapy and computed tomography (CT) imaging. AuNPs absorb x-rays and subsequently release low-energy, short-range photoelectrons during external beam radiation therapy (RT), increasing the local radiation dose. When AuNPs are near tumor vasculature, the additional radiation dose can lead to increased vascular permeability. This work focuses on understanding how tumor vascular permeability is influenced by AuNP-augmented RT, and how this effect can be used to improve the delivery of nanoparticle chemotherapeutics. Methods: Dual-energy CT was used to quantify the accumulation of both liposomal iodine and AuNPs in tumors following AuNP-augmented RT in a mouse model of primary soft tissue sarcoma. Mice were injected with non-targeted AuNPs, RGD-functionalized AuNPs (vascular targeting), or no AuNPs, after which they were treated with varying doses of RT. The mice were injected with either liposomal iodine (for the imaging study) or liposomal doxorubicin (for the treatment study) 24 hours after RT. Increased tumor liposome accumulation was assessed by dual-energy CT (iodine) or by tracking tumor treatment response (doxorubicin). Results: A significant increase in vascular permeability was observed for all groups after 20 Gy RT, for the targeted and non-targeted AuNP groups after 10 Gy RT, and for the vascular-targeted AuNP group after 5 Gy RT. Combining targeted AuNPs with 5 Gy RT and liposomal doxorubicin led to a significant tumor growth delay (tumor doubling time ~ 8 days) compared to AuNP-augmented RT or chemotherapy alone (tumor doubling time ~3-4 days). Conclusions: The addition of vascular-targeted AuNPs significantly improved the treatment effect of liposomal doxorubicin after RT, consistent with the increased liposome accumulation observed in tumors in the imaging study. Using this approach with a liposomal drug delivery system can increase specific tumor delivery of chemotherapeutics, which has the potential to significantly improve tumor response and reduce the side effects of both RT and chemotherapy.

Mice Lacking RIP3 Kinase are not Protected from Acute Radiation Syndrome

Exposure to high doses of ionizing radiation can cause lethal injury to normal tissue, thus inducing acute radiation syndrome. Acute radiation syndrome is caused by depletion of bone marrow cells (hematopoietic syndrome) and irreparable damage to the epithelial cells in the gastrointestinal tract (gastrointestinal syndrome). Although radiation initiates apoptosis in the hematopoietic and gastrointestinal compartments within the first few hours after exposure, alternative mechanisms of cell death may contribute to injury in these radiosensitive tissues. In this study, we utilized mice lacking a critical regulator of necroptosis, receptor interacting protein 3 (RIP3) kinase, to characterize the role of RIP3 in normal tissue toxicity after irradiation. Our results suggest that RIP3-mediated signaling is not a critical driver of acute radiation syndrome.

Characterizing the potency and impact of carbon ion therapy in a primary mouse model of soft tissue sarcoma

Carbon ion therapy (CIT) offers several potential advantages for treating cancers compared with X-ray and proton radiotherapy, including increased biological efficacy and more conformal dosimetry. However, CIT potency has not been characterized in primary tumor animal models. Here, we calculate the relative biological effectiveness (RBE) of carbon ions compared with X-rays in an autochthonous mouse model of soft tissue sarcoma. We used Cre/loxP technology to generate primary sarcomas in KrasLSL-G12D/+; p53fl/fl mice. Primary tumors were irradiated with a single fraction of carbon ions (10 Gy), X-rays (20 Gy, 25 Gy, or 30 Gy), or observed as controls. The RBE was calculated by determining the dose of X-rays that resulted in similar time to posttreatment tumor volume quintupling and exponential growth rate as 10 Gy carbon ions. The median tumor volume quintupling time and exponential growth rate of sarcomas treated with 10 Gy carbon ions and 30 Gy X-rays were similar: 27.3 and 28.1 days and 0.060 and 0.059 mm3/day, respectively. Tumors treated with lower doses of X-rays had faster regrowth. Thus, the RBE of carbon ions in this primary tumor model is 3. When isoeffective treatments of carbon ions and X-rays were compared, we observed significant differences in tumor growth kinetics, proliferative indices, and immune infiltrates. We found that carbon ions were three times as potent as X-rays in this aggressive tumor model and identified unanticipated differences in radiation response that may have clinical implications. Mol Cancer Ther; 17(4); 858–68. ©2018 AACR.

Abstract LB-167: Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Radiation therapy is frequently utilized in the clinic as curative treatment for cancers, but the contribution of endothelial cells to tumor response to radiation remains controversial. Using the two highly efficient site-specific recombinases, Cre and FlpO, we have generated primary tumors in mice with different gene mutations specifically in tumor cells and stromal cells. With this dual recombinase technology, we selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. We found that deletion of Bax in endothelial cells did not affect tumor response to radiation, but Atm deletion increased radiation-induced death of tumor endothelial cells and prolonged tumor growth delay following a non-curative dose of radiation. However, following a curative dose of radiation, Atm deletion in endothelial cells did not affect growth delay of primary tumors and failed to increase local control. In contrast, deletion of Atm in tumor cells increased local control of primary tumors by radiation therapy. These results demonstrate that tumor cells rather than endothelial cells are the critical targets that regulate tumor eradication by radiation therapy. They also emphasize the importance of using primary models of cancer to study the role of stromal cells in tumor development and response to therapy. Citation Format: Everett J. Moding, Chang-Lung Lee, Katherine D. Castle, Patrick Oh, David G. Kirsch. Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2014-LB-167