Combining inotuzumab ozogamicin with the p38 inhibitor BIRB 796 and venetoclax exerts synergistic cytotoxicity in B-cell precursor acute lymphoblastic leukemia including very high risk t(17;19) acute lymphoblastic leukemia

To improve mechanism-based pharmacotherapy for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), we analyzed mode of action and potential synergies of inotuzumab ozogamicin (INO) with the p38 kinase inhibitor BIRB 796 and the BCL2-specific BH3 mimetic venetoclax (VEN) in BCP-ALL cell lines and patient-derived xenograft (PDX) models including t(17;19) PDX cells. INO is an antibody-drug conjugate (ADC) consisting of an anti-CD22 antibody linked to calicheamicin (CAL). CAL and INO induced DNA strand cleavage and activated DNA damage response signaling, including phosphorylation of ATM, H2AX (γH2AX) and p38, as well as induction of p53 protein expression. Pharmacologic inhibition of p38 with BIRB 796 selectively potentiated INO-induced DNA strand cleavage, increased γH2AX expression and enhanced cytotoxicity, whereas CAL activity remained unaffected in line with drug-interaction upstream of DNA damage. Furthermore, INO induced mitochondrial priming and augmented VEN-mediated mitochondrial outer membrane permeabilization, which was further enhanced by BIRB 796. Accordingly, INO and VEN exerted synergistic cytotoxicity in the presence and absence of BIRB 796. Finally, this triple therapy induced complete remissions assessed by bioluminescence imaging and long-term leukemia-free and overall survival in 5 out of 7 (71%) NSG mice engrafted with L707 (t(17;19)) PDX cells. These findings identify time-limited p38 inhibition as a mechanism-based strategy to enhance INO-induced DNA cleavage and mitochondrial priming, resulting in markedly improved therapeutic efficacy in a very high-risk leukemia model. This work may provide a rationale for optimizing ADCbased combination therapies through transient inhibition of p38.