Off-Target MAP2K7 Inhibition Drives the Antileukemic Activity of the PD-1/PD-L1 inhibitor ACS-69 in Pediatric T-ALL

<title>Abstract</title> <p> T-cell acute lymphoblastic leukemia (T-ALL) remains a high-risk pediatric malignancy characterized by aggressive disease behavior and poor outcomes after relapse, underscoring the need for novel targeted therapies. Our group reported that aberrant activation of the MAP2K7–JNK signaling axis is implicated in T-ALL pathogenesis, promoting leukemic cell survival, proliferation, and treatment resistance. In this study, we evaluated the anti-leukemic properties of the compound ASC69 in T-ALL cell lines and patient-derived xenograft cells using cell viability, apoptosis, and cell cycle analyses. Pathway inhibition was examined by immunoblotting. ASC69 exhibited dose-dependent cytotoxicity across multiple T-ALL cell lines, with IC₅₀ values in the low nanomolar range, while showing reduced cytotoxicity against normal human bone marrow cells. Genetic ablation of the MAP2K7 gene in Jurkat cells significantly increased the ASC69 IC ₅₀ , supporting MAP2K7 as the primary functional target. Mechanistically, ASC69 induced G2/M cell-cycle arrest and apoptosis, as determined by Annexin V staining and cleavage of PARP and Caspase-3. <italic>In vitro</italic> kinase assays demonstrated direct inhibition of MAP2K7 enzymatic activity across multiple activation conformations, including wild-type, an active phosphomimetic mutant, and an active phosphomimetic mutant with a 75-deletion in the N-terminus. Consistent with on-target pathway engagement, ASC69 inhibited both basal and stress-induced phosphorylation of JNK and ATF2 in T-ALL cell lines. Importantly, ASC69 demonstrated cytotoxic activity in T-ALL patient-derived xenograft cells and synergized with standard-of-care T-ALL drugs. Collectively, these findings establish ASC69 as a promising MAP2K7-targeted therapeutic candidate and support preclinical studies in T-ALL models. </p>