Plinabulin and Selumetinib Enhance M1 Macrophage Traits and Trigger Cell Death in AML
Authors: Pinheiro Lucio Henrique , Weinhäuser Isabel , Pereira-Martins Diego A. , Simoes Luise Araujo De Albuquerque , Markham Matthew , Quek Lynn , Huls Gerwin , Schuringa Jan Jacob , Rushworth Stuart A , Rego Eduardo M. 
Published: December 2024

Acute myeloid leukemia (AML) exhibits significant heterogeneity, which also extends to its tumor microenvironment (TME). Our group showed that macrophages associated with AML (AAMs) often exhibit an M2-like phenotype, characterized by a unique molecular program (M2-signature) linked to poor therapeutic response and prognosis (Weinhäuser et al., Sci Advances 2023-doi.org/10.1126/sciadv.adf8522). In vivo studies show that AAMs support leukemic proliferation by rewiring the metabolism of leukemic blasts towards oxidative phosphorylation, thereby circumventing the phagocytosis barriers of the BM niche and promoting a pro-leukemic environment. Conversely, M1 macrophages demonstrated anti-tumorigenic properties suggesting that the therapeutic repolarization of AAMs into M1 macrophages could be beneficial in AML treatment. Using our M2-signature in a larger cohort of ex vivo treated AML samples (n=157, BeatAML study) and a 127-drug panel, we identified that selumetinib (AZD6244, MEK1/2 inhibitor) and drugs targeting microtubule biology are effective in M2high AMLs. Plinabulin (BPI-2358), a tubulin polymerization blocker, has been reported to promote M1-like polarization and exhibit anti-tumor activity (Natoli et al., Front. Oncol 2021 - doi.org/10.3389%2Ffonc.2021.644608). We then investigated the ability of AZD6244 and BPI-2358 to repolarize macrophages in vitro. Unpolarized healthy human peripheral blood (PB)-derived M0 macrophages were treated for 48 hours with either BPI-2358 (0.125-0.5 µM) and AZD6244 (0.25-1 µM). Immunophenotypic analysis revealed that treatment with both drugs increased the expression of M1 markers CD80 and CD86 while reducing the M2 marker MRC1 (CD206). In murine BM-derived macrophages (from 10-week-old PEPC-Boy mice, 50% males), similar treatments increased the gene expression of M1 markers Cd80 and TNFα and decreased the M2 marker Arg1 in a dose-dependent manner. These findings suggest that both BPI-2358 and AZD6244 enhance pro-inflammatory features in M2 macrophages, potentially contributing to anti-tumor immune surveillance. To evaluate the potential of these drugs to repolarize tumor-associated macrophages (TAMs), we co-cultured unpolarized BM-derived murine M0 macrophages with murine AML models created by overexpressing MN1 and MEIS/HOXA9 (Moore et al., JCI 2022 - doi.org/10.1172/jci153157). After 72 hours of co-culture, the tumor cells were removed, and the TAMs were treated with BPI-2358 (0.5 µM) and AZD6244 (1 µM) for 48 hours. BPI-2358 treatment led to increased expression of both the M1 marker Cd80 and the M2 marker Mrc1 (Cd206) in both TAM models. AZD6244 treatment resulted in increased Cd80 expression only in the MN1-TAM model, while it increased Mrc1 expression in both models. To further assess the drugs anti-leukemic activity and ability to repolarize AAMs, we conducted an ex vivo drug screening using a panel of genetically diverse AML samples (n=10). Treatment with BPI-2358 (0.125-0.5 µM) and AZD6244 (0.25-1 µM) for 72 hours showed significant effects on leukemic blasts (CD34+ or CD117+ for CD34- AMLs) and AAMs (CD34- or CD117-HLADR+CD14+), significantly reducing CD163 levels in the AAM population, indicating a preference for targeting M2-like macrophages in AML. Additionally, both drugs were associated with reduced mitochondrial membrane potential in leukemic blasts and AAMs, suggesting an impact on mitochondrial metabolism. In conclusion, we found that both BPI-2358 and AZD6244 exhibit potential in repolarizing M2-like TAMs towards an M1-like phenotype, and have cytotoxic activity in AAM and leukemic blasts thus representing a promising avenue for enhancing AML treatment strategies.