A combination strategy of inotuzumab ozogamicin (InO) and BCL-2 family inhibitors prevents the emergence of PGP+ ino-resistant MRD in B-cell acute lymphoblastic leukemia

Abstract Background: Inotuzumab ozogamicin (InO) achieves a complete remission (CR) rate of over 60-80% in patients with relapsed/refractory B cell-lineage acute lymphoblastic leukemia (r/r B-ALL), however durable remissions are rare. Relapse is presumably due to the persistence and regrowth of minimal residual disease (MRD) that has acquired InO resistance. Aim and Method: We aimed to elucidate the molecular mechanisms of InO resistance by analyzing the protein expression profile of residual leukemic blasts from clinical samples, experimental cell-based and mouse models, with the goal of developing a strategy to eliminate these MRD cells. Results: Among patients who achieved hematological CR following InO treatment (n=13) in the AALL1621 phase 2 trial, immunophenotypic analysis using B cell-lineage markers detected 0.01-1.0% residual bone marrow blasts. Cell-community clustering revealed that these MRD populations were predominantly composed of Pgp+/Bcl-2+ cells (P-glycoprotein, encoded by ABCB-1) retaining CD22 expression; these cells were not detected prior to InO treatment but emerged afterward and persisted beyond the end of cycle 2. These findings suggest that InO monotherapy can induce the emergence of MRD cells that newly acquire InO-resistant features such as Pgp expression. Consistent with these clinical findings, in vitro experiments using B-ALL cell lines such as NALM6 and REH showed that InO treatment led to persistent upregulation of Bcl-2 and p53 proteins while retaining CD22, ultimately resulting in the emergence of Pgp+, InO-refractory cells (InO-R/Pgp+ ALL cells). To confirm the role of Pgp, we generated ABCB1-transduced ALL cells and assessed the sensitivity to InO. These cells exhibited complete resistance to InO, indicating that Pgp-mediated drug efflux can override CD22-targeted cytotoxicity by InO. Intriguingly, InO-R/Pgp+ ALL cells were highly sensitive to Bcl-2 inhibitors, showing marked killing effects, whereas ABCB1-transduced ALL cells were far less responsive. The RNA sequencing of InO-R/Pgp+ ALL cells revealed significant activation of the p53 pathway and upregulation of pro-apoptotic BH3-only proteins and effector molecules (e.g., PUMA, BAX). These findings suggest that the survival of InO-R/Pgp+ ALL cells is highly dependent on the anti-apoptotic activity of the Bcl-2 family, thereby rendering the cells highly sensitive to Bcl-2 inhibitors despite resistance to InO. We next evaluated the synergistic effects of InO and Bcl-2 family inhibitors to prevent the emergence of InO-R/Pgp+ ALL cells. Venetoclax (VEN) and navitoclax, not S63845 (Mcl1 inhibitor) synergized with InO to enhance the killing of ALL cells. In a cell-derived xenograft model, a single cycle of InO and VEN combination therapy achieved a more prolonged remission than InO alone. Notably, Pgp expression was observed on leukemic cells from mice that relapsed after InO monotherapy, but not in those that received the combination therapy. Conclusion: While InO therapy achieves high remission rates, it may induce the emergence of InO-R/Pgp+ ALL cells as residual disease. Combination therapy with InO and Bcl-2 family inhibitors may effectively suppress the emergence of these InO-resistant clones, thereby prolonging remission duration.