Abstract 1058: Evaluation of a novel class of bifunctional DNA alkylating agent and PARP inhibitor
Authors: Truong Sarah , Zhai Beibei , Ghaidi Fariba , Ramos Louise , Joshi Jay , Brown Dennis , Sankar Neil , Langlands John , Bacha Jeffrey , Shen Wang , Daugaard Mads 
Published: June 2022

Abstract
Introduction: Poly-adenosine diphosphate-ribose polymerase (PARP) inhibition is an effective, FDA-approved treatment against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 or BRCA2 mutations. PARP is a core component of single-strand break repair, so in tumors with DDR deficiencies, double-strand breaks (DSBs) and single-strand breaks (SSBs) accumulate and lead to cell death. Combining PARP inhibition with other DNA-damaging therapies, such as DNA alkylating agents, has proven to be more effective than PARP inhibition alone, as DNA alkylators can lead to the formation of DSBs. However, combination therapies often require sequential administration due to pharmacokinetic considerations and overlapping toxicities, severely limiting their clinical utility. Here, we evaluate a novel class of PARP inhibitors combined with DNA alkylating functionality, in formulation as a single molecule.
Methods: PARP1 activity was determined using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was determined using the BPS Bioscience PARP2 Colorimetric Assay Kit. Methylator release was evaluated by measuring the appearance of a certain degradation product. Cell cycle analysis was performed using propidium iodide staining and quantified using flow cytometry. pH2AX expression as a measure of DNA double strand breaks (DSB) was quantified by flow cytometry using an Alexa Fluor 647-conjugated anti-H2AX phospho (Ser139) antibody from BioLegend. Detection of pH2AX by western blot and immunofluorescence assays was done using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies.
Results: kt-4000 series compounds are potent inhibitors of both PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compounds release methylator in a similar manner as temozolomide, an FDA-approved DNA alkylating agent. They induce S-phase and G2/M cell cycle arrest in MCF7 cells and lead to accumulation of pH2AX by flow cytometry, western blot, and immunofluorescence assays.
Conclusion: Our novel class of PARP inhibitors combined with DNA alkylating function show potent inhibition of both PARP1 and PARP2. These compounds also lead to accumulation of H2AX phosphorylation, a sensitive marker for double-strand breaks, and S-phase and G2/M cell cycle arrest. Development of these bifunctional, single molecule therapies may extend the clinical utility of PARP inhibitors to DDR proficient disease and increase efficacy for DDR deficient disease.
Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Mads Daugaard. Evaluation of a novel class of bifunctional DNA alkylating agent and PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1058.