AML-269: The Anti-Leukemic Effects of the Pan-Aurora Inhibitor Tozasertib Are Negatively Influenced by Hypoxia in Acute Myeloid Leukemia

Abstract

<h3>Context:</h3> The hypoxic bone marrow microenvironment plays a crucial role in acute myeloid leukemia (AML) development and progression, acting as a sanctuary for leukemic stem cells (LSCs). Hypoxia drives transcriptomic and metabolomic changes that enhance the fitness of LSCs, reducing their susceptibility to drug treatment. Aurora kinases A and B are crucial actors during mitosis and their overexpression has been described in AML, making them promising therapeutic targets. Phase II studies of Aurora kinase inhibitors combined with induction chemotherapy have demonstrated good tolerability and efficacy in patients with high-risk AML. <h3>Objective:</h3> We aimed to investigate the influence of hypoxia on AML sensitivity to Aurora kinase inhibition. <h3>Materials:</h3> AML cell lines (NOMO-1: t(9;11), p53 mutated; MOLM-13: FLT3-ITD; MV-4-11: t(4;11), +8, +19, FLT3-ITD; KASUMI-1 t(8;21), c-KIT and p53 mutated) were treated under either normoxia or hypoxia (1% O₂) with the pan-Aurora inhibitor tozasertib (100 and 250 nM for 24 and 48 hours). Annexin V and propidium iodide (PI) staining were performed to evaluate the induction of apoptosis and proliferation capacity. Protein level alterations were analyzed by Western blotting (WB). <h3>Results:</h3> Hypoxia strongly influenced the anti-leukemic effects of tozasertib, reducing its ability to inhibit growth and proliferation in AML cells. In particular, all tested AML cell lines cultured under hypoxia showed reduced sensitivity to tozasertib compared to those under normoxia, with a significant reduction of apoptotic death after 48h of treatment. AnnexinV+ cells ranged between 32 and 60% under hypoxia <i>vs</i> 47-70% under normoxia (p<0.05). Moreover, hypoxic culture conditions significantly reduced the ability of tozasertib to induce cell cycle arrest in G2/M phase already after 24 hours of treatment. Inhibition of Aurora kinases was associated with activation of the TP53 pathway, inducing the expression of phospho-p53 (ser15), p21 and γH2AX as well as the cleavage of caspase 3 and PARP in all tested cell lines. However, the up-regulation of the above proteins was less pronounced under hypoxic conditions. <h3>Conclusion:</h3> Our study shows that, irrespective of the genetic background, cellular responses to Aurora inhibition in AML are markedly impaired by hypoxia. This suggests that AML LSCs might not be eradicated by tozasertib treatment.