Inhibiting tyrosine phosphorylation of protein kinase Cδ (PKCδ) protects the salivary gland from radiation damage.

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of "gate-keeper" mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.

Tyrosine Kinase Inhibitors Protect the Salivary Gland from Radiation Damage by Inhibiting Activation of Protein Kinase C-δ.

In patients undergoing irradiation (IR) therapy, injury to nontumor tissues can result in debilitating, and sometimes permanent, side effects. We have defined protein kinase C-δ (PKCδ) as a regulator of DNA damage-induced apoptosis and have shown that phosphorylation of PKCδ by c-Abl and c-Src activates its proapoptotic function. Here, we have explored the use of tyrosine kinase inhibitors (TKI) of c-Src and c-Abl to block activation of PKCδ for radioprotection of the salivary gland. Dasatinib, imatinib, and bosutinib all suppressed tyrosine phosphorylation of PKCδ and inhibited IR-induced apoptosis in vitro To determine whether TKIs can provide radioprotection of salivary gland function in vivo, mice were treated with TKIs and a single or fractionated doses of irradiation. Delivery of dasatinib or imatinib within 3 hours of a single or fractionated dose of irradiation resulted in >75% protection of salivary gland function at 60 days. Continuous dosing with dasatinib extended protection to at least 5 months and correlated with histologic evidence of salivary gland acinar cell regeneration. Pretreatment with TKIs had no impact on clonogenic survival of head and neck squamous cell carcinoma (HNSCC) cells, and in mice harboring HNSCC cell-derived xenografts, combining dasatinib or imatinib with fractionated irradiation did not enhance tumor growth. Our studies indicate that TKIs may be useful clinically to protect nontumor tissue in HNC patients undergoing radiotherapy, without negatively impacting cancer treatment. Mol Cancer Ther; 16(9); 1989-98. ©2017 AACR.