Azeliragon has significant activity against acute myeloid leukemia blasts

Abstract High Mobility Group Box-1 (HMGB1) is a non-histone chromosome binding protein that has a dual function. Intracellular HMGB1 binds to DNA and is involved in transcriptional regulation, DNA replication, DNA repair, telomere maintenance, and nucleosome assembly. HMGB1 can also be passively released by necrotic or stressed cells, or actively secreted. When secreted, HMGB1 acts as a chemokine playing the role of a Damage-Associated Molecular Pattern (DAMP). As a DAMP, HMGB1 is involved in inflammatory response and signals through multiple receptors, including the Receptor for Advanced Glycation End Products (RAGE). Autophagy is a self-protective process that promotes cell survival under stress conditions. Autophagy degrades long-lived proteins, damaged organelles and abnormal protein aggregates. Autophagy induces cancer cell drug resistance, including in leukemia cells. When released by dying cancer cells, HMGB1 can signal through RAGE to induce autophagy in adjacent cells, potentially inducing resistance to chemotherapy. Extracellular HMGB1 was shown to interact with RAGE to induce autophagy and inhibit apoptosis in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells. This was associated with activation of ERK1/2 and decreased phosphorylation of mTOR. In addition, the anti-apoptotic effect of HMGB1 was associated with upregulation of bcl2, an effect that was RAGE-dependent. Azeliragon (AZE) is an orally available small molecule inhibitor of RAGE-ligand binding. It initially entered human clinical trials for the treatment of Alzheimer's disease where it was well tolerated. AZE is currently in clinical trials for the treatment of solid tumors. The goal of this study was to investigate the effect of AZE on AML cell lines (HL-60 and OCI-AML3) and patient-derived AML cells. HL-60 and OCI-AML3 were cultured in RPMI-1640 with 10% fetal bovine serum. Patient-derived cells were cultured in SFEM II, CD34 Expansion Supplement, and UM729. Patient-derived cells (4 from peripheral blood, 1 from bone marrow aspirate) were plated within 18 hours of collection and cultured for 1-3 days prior to addition of AZE. One additional patient-derived sample was collected by leukapheresis and cultured for 24 days to expand the rare blast population before adding AZE. Cells were incubated for 72 hours with 0-10 µM of AZE, before analysis by MTS (cell lines) or CellTiter Glo (patient cells). HL-60 and OCI-AML3 cell growth were completely eradicated by 3 µM AZE, with IC50 values of 2.2 µM and 1.7 µM, respectively. Synergy analysis of OCI-AML3 cells with Combenefit software demonstrated high synergy scores when AZE was combined with cytarabine or venetoclax, medium synergy scores when combined with daunorubicin, and antagonism when combined with azacitidine. HL60-cells showed no consistent synergy or antagonism signal across the three models in the Combenefit analysis with any drug combination. Blast-enriched samples from 6 AML patients (3 de novo untreated, 3 relapse) were incubated with 0-10 µM AZE. Cells were completely eradicated by 7.5-10 µM AZE (IC50=2-7 µM). The relapse patients included 1 with relapse after 2 bone marrow transplants, 1 with relapse after 7 lines of treatment (including Revumenib), and another 1 with relapse after Revumenib. AZE was effective against AML cells with KMT2A point mutations, duplications, gains, and rearrangements, suggesting a potential treatment option for patients after the use of menin inhibitors. Synergy analysis indicated synergy scores >30 with ≤5 µM AZE combined with 10 nM venetoclax and 0.75 µM azacitidine for 5/6 samples. The remaining sample was eradicated at 7.5 µM AZE, with a synergy score of 10 at 3.75 µM AZE. We conclude that AZE shows promising single agent activity in AML with potential for synergistic combinations with standard agents. Clinical investigation of AZE for the treatment of AML is warranted.