Abstract 541: Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming drug resistance
Authors: Ida Naoko , Okura Miyuki , Hosono Naoko , Yamauchi Takahiro 
Published: March 2024

Abstract
Introduction: Inotuzumab Ozogamicin (IO) is a humanized anti-CD22 monoclonal antibody conjugated with chalicheamicin, which causes single- and double-strand DNA breaks. Although IO shows a high CR/CRi rate in relapsed/refractory CD22-positive B-cell acute lymphoblastic leukemia (ALL), the duration of remission is short due to early acquisition of resistance. Therefore, the present study attempted to enhance the cytotoxicity of IO by focusing on polyadenosine diphosphate-ribose polymerase (PARP) inhibition. Furthermore, IO-resistant cell lines were newly established to investigate the mechanism of IO resistance.
Methods: Reh cell line derived from Ph-negative B-ALL cells was used. To establish IO-resistant cell lines, Reh cells were long-term exposed to IO, followed by limiting dilution. FACS analysis evaluated the CD22 positivity and the induction of apoptosis. WTS assay determined the cell growth inhibition effects. The Comet assay quantitatively assessed DNA damage. Gene expression profiles were determined by using DNA microarray.
Results: The 50%-inhibitory concentration (IC50) of IO was 2.1 ng/mL in Reh cells. Reh cells were synergistically sensitized to IO by the addition of non-toxic concentrations of PARP inhibitors, olaparib or talazoparib, with the Combination Index of 0.19 and 0.42, respectively. Moreover, these combinations augmented the induction of apoptosis (23.5% in IO alone, 51.7% in IO + olaparib, 66.1% in IO + talazoparib). The Comet assay demonstrated that DNA strand breaks observed 1h after IO administration were repaired 6h later. In the combination of IO with olaparib or talazoparib, DNA damage remained even at 6h, suggesting the inhibition of DNA repair by PARP inhibitors. Next, six IO-resistant cell lines were established with IC50 more than 60-fold that of parental cells. All these cell lines kept CD22 expression. These cell lines were also resistant to apoptosis induced by IO. In addition of olaparib or talazoparib to IO, the IC50 of IO decreased by 40% to 70% in 6 IO-resistant cells. These combinations also augmented the induction of apoptosis (24.2% in IO alone, 45.3% in IO+olaparib, and 56.8% in IO+talazoparib). DNA microarray was performed to compare gene expression profiles between parental and IO-resistant cell lines. ABCB1 (p-gp), a drug efflux pump that causes multi-drug resistance, was overexpressed in resistant cell lines. Moreover, the addition of p-gp inhibitor, PSC-833, to IO enhanced the cell growth inhibition and induction of apoptosis due to more DNA strand break.
Conclusion: The present study demonstrated that the combination of IO with PARP inhibitors exerted synergistic cytotoxicity via the inhibition of DNA repair. In addition, it was suggested that overexpression of p-gp caused IO resistance, which was partially reversed with the combination of PARP inhibitors or a p-gp inhibitor.
Citation Format: Naoko Ida, Miyuki Okura, Naoko Hosono, Takahiro Yamauchi. Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 541.