Signaling Network Response to Alpha-Particle Targeted Therapy with Actinium-225 Labeled Minigastrin Analogue 225Ac-PP-F11N Reveals Radiosensitizing Potential of HDAC Inhibitors.

Alpha-particle emitters have recently been explored as valuable therapeutic radionuclides. Yet, toxicity to healthy organs and cancer radioresistance limit the efficacy of targeted alpha-particle therapy (TAT). Identification of the radiation-activated mechanisms, which drive cancer cell survival, provides opportunities to develop new points for therapeutic interference to improve efficacy and safety of TAT. Methods: Quantitative phosphoproteomics and matching proteomics followed by the bioinformatics analysis were employed to identify alterations in the signaling networks in response to TAT with actinium-225 labeled minigastrin analogue 225Ac-PP-F11N in A431 cells, which overexpress cholecystokinin B receptor (CCKBR). Western blot (WB) analysis and microscopy verified the activation of the selected signaling pathways. Small-molecule inhibitors were used to validate the potential of the radio-sensitizing combinatory treatments both in vitro and in A431/CCKBR tumor-bearing nude mice. Results: TAT-induced alterations involved in DNA damage response (DDR), cell cycle regulation, signal transduction as well as RNA transcription and processing, cell morphology and transport. WB analysis and microscopy confirmed increased phosphorylations of the key proteins involved in DDR and carcinogenesis including P53, P53BP1 histone deacetylases (HDACs) and H2AX. Inhibition of HDAC class II, ataxia-telangiectasia mutated (ATM) and p38 kinases by TMP269, AZD1390 and SB202190, respectively, sensitized A431/CCKBR cells to 225Ac-PP-F11N. Combination of 225Ac-PP-F11N with HDAC inhibitor vorinostat (SAHA) showed significantly reduced viability and increased DNA damage of A431/CCKBR cells as well as the most pronounced tumor growth inhibition and the extended mean survival of A431/CCKBR xenografted nude mice as compared to the control and monotherapies. Conclusion: Our study revealed the cellular responses to TAT and demonstrated the radiosensitizing potential of HDAC inhibitors to 225Ac-PP-F11N in CCKBR-positive tumors. This proof-of-concept study recommends development of the novel radiosensitizing strategies by targeting TAT-activated and survival-promoting signaling pathways.