Investigation of ATR Inhibitor VX970 as a Radiosensitizer in Colorectal Cancer Cells

Abstract

Colorectal cancer (CRC) is the second leading cause of combined cancer-related mortality in males and females in the U.S. Traditional treatment of locally advanced rectal cancer consists of neoadjuvant chemoradiation followed by surgery and adjuvant chemotherapy. Emerging data suggests that higher response rates can be achieved with total neoadjuvant therapy (TNT) where delivery of all chemotherapy and radiation therapy (RT) occurs prior to surgery. In addition, for patients with a complete response to TNT, non-operative management (NOM) can be considered. However, despite the use of TNT, pathologic complete response rates remain below 40% and NOM is only achieved in approximately 50% of rectal cancer patients. A strong need remains for more active anti-cancer therapies in rectal cancer to both reduce pelvic recurrence and facilitate NOM. Here, we tested the hypothesis that inhibition of the ataxia telangiectasia and Rad3-related protein kinase (ATR), a critical regulator of cellular DNA damage response, could increase the sensitivity of CRC to RT. VX970, a highly potent and selective ATR inhibitor, was investigated as a radiosensitizer in SW48 and LoVo CRC cell models. In vitro, IC50 of VX970 was assessed by alamarBlue cytotoxicity assay, while radiosensitivity was revealed by radiation clonogenic assays (0, 2, 4, 6, 8 Gy). ATR activity was determined by p-Chk1 using immunoblotting, and cell cycle distribution was analyzed by propidium iodide flow cytometry. CRC xenografts were generated using both LoVo and SW48 cells injected in the left flanks of athymic nude mice to explore the radiosensitizing effects of VX970 in vivo. Tumors were allowed to grow to 100-150 mm3, and the mice were randomized into multiple groups [vehicle alone, RT alone (10 Gy/5 fractions), VX970 alone, and VX970+RT]. Mouse weights and tumor size were measured three times weekly. Comparison of treatment groups was performed using the log-rank test with P<0.05 considered significant. The IC50 concentrations of VX970 on SW48 and LoVo cells were about 500 and 100 nM, respectively. VX970 at doses of 3 nM did not alter the viability of CRC cells, but significantly sensitized CRC cells to radiotherapy (P<0.05), with DER of 1.43 and 1.59, respectively, in SW48 and LoVo cells. VX970 efficiently attenuated p-Chk1 expression and significantly abrogated radiation induced G2/M cell cycle arrest (P<0.05). In addition, VX970 in combination with radiotherapy significantly prolonged tumor growth delay of CRC xenografts compared to radiation alone (P<0.05), with minimal toxicity observed. Inhibition of the ATR-Chk1 pathway by targeting ATR kinase with VX970 sensitizes CRC to radiotherapy in vitro and in vivo. Our findings support that ATR inhibition by VX970 is a promising new approach to improve the therapeutic ratio of radiotherapy for patients with CRC and warrants further clinical testing.