Abstract 433: Clarification and overcoming the resistance mechanisms to inotuzumab ozogamicin and ponatinib in B-ALL

Abstract [Introduction] Recently, the prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has significantly improved with the appearance of novel drugs, including inotuzumab ozogamicin (IO) and ponatinib. IO is an anti-CD22 antibody conjugated with calicheamicin and highly effective for B-ALL. Ponatinib, a third-generation tyrosine kinase inhibitor for Philadelphia chromosome (Ph)-positive ALL. The acquisition of resistance to these latest drugs is a major clinical hurdle. In the previous study, we showed that the addition of polyadenosine diphosphate-ribose polymerase (PARP) inhibitors synergistically enhanced the cytotoxicity of IO via the inhibition of the leukemic DNA damage repair mechanism. The present research attempted to clarify and overcome the resistance mechanism to IO and ponatinib using newly established drug-resistant cell lines. [Method] Gene expression were analyzed using DNA microarray. Protein expression was analyzed by western blotting. Quantitation of DNA damage and repair was measured using the alkaline comet assay. Additional aberrations of Ph were examined by G-banding and FISH analysis. BCR::ABL point mutations were analyzed by direct sequencing. [Results] We successfully established 6 IO-resistant cell lines that were 60-fold more resistant to IO than the parental cell line. All 6 cell lines were also resistant to apoptosis. In resistant cells, DNA microarray showed that ABCB1 gene encoding p-gp, drug efflux pump, was overexpressed, and western blotting revealed the overexpression of p-gp protein. The combination of IO and a p-gp inhibitor, PSC-833, decreased the IC50 values of IO by 90% and increased apoptosis. Alkaline comet assay revealed increased DNA damage in the combination. When combined with IO and PARP inhibitors, olaparib and talazoparib, increased the IO sensitivity of resistant cell lines by 50 to 90%. The triple combination of p-gp inhibitor, PARP inhibitors and IO improved the IO sensitivity to the same level as the parental cell line. The alkaline comet assay indicated DNA damage observed at 1 hour after administration of IO and restored after 6 hours. However, in the triple combination, DNA damage that was observed 1 hour more increased after 6 hours. In the ponatinib-resistant cell line, the IC50 value of the ponatinib was 100-fold higher than in the parental cell line. No additional chromosomal abnormalities other than Ph were detected in G-banding in the ponatinib-resistant cell line. The BCR::ABL breakpoint was the same , and no point mutations were detected in parental and resistant cell lines. DNA microarray showed that JAK2 expression was upregulated in resistant cell line. [Conclusion] The present study indicated that inhibition of drug efflux and DNA damage repair overcame the resistance to IO in Ph-negative B-ALL. Moreover, JAK2 might be the therapeutic target to overcome the resistance to ponatinib in Ph-positive B-ALL. Citation Format: Naoko Ida, Miyuki Okura, Naoko Hosono, Takahiro Yamauchi. Clarification and overcoming the resistance mechanisms to inotuzumab ozogamicin and ponatinib in B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 433.