BPI-2358 reprograms immunosuppressive macrophages and impairs leukemic progression in MN1-overexpressing AML In Vivo

Abstract

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by the accumulation of immature myeloid cells in the bone marrow (BM) and peripheral blood (PB). Several alterations in the BM microenvironment have been linked to AML onset and progression, some of which affect the immune system and promote tumor evasion. Genetic alterations also contribute to these processes, including overexpression of the MN1 gene, which is addressed in this study. MN1 overexpression is associated with a more aggressive leukemic phenotype and therapeutic resistance. Among the pathological findings in the leukemic BM microenvironment, the relative increase in AML-associated macrophages (AAMs) appears to support AML progression. AAMs display a phenotype similar to that of healthy M2-like macrophages and are thus linked to anti-inflammatory and immunosuppressive activity. In this context, therapeutic approaches aimed at reprogramming AAMs toward a more inflammatory and antitumoral profile, resembling M1-like macrophages, have been investigated. This study aimed to evaluate the immunomodulatory effects of BPI-2358, a tubulin polymerization inhibitor and JNK pathway activator, on AAMs in AML models driven by MN1 overexpression. Human PB-derived M2 macrophages were treated with BPI-2358 (0.125–0.5 µM) for 48 hours. Immunophenotyping (CD80, CD86, CD206, CD163) and cytokine analysis (TNF-α, IL-1β, TGF-β) revealed a shift toward a pro-inflammatory profile. Our findings showed that BPI-2358 treatment had a significant impact on marker expression, leading to an upregulation of CD86 and a downregulation of CD163 in human samples. Additionally, we detected elevated levels of pro-inflammatory cytokines, including IL-1β and TNF-α, along with a reduction in TGF-β secretion. Similarly, murine BM-derived M2 macrophages treated under the same conditions showed increased expression of M1-related genes (Cd80, Tnf-α, Nos2) and downregulation of Arg1, a classical M2 marker, by qRT-PCR. A phagocytosis assay was performed using M2-polarized murine macrophages treated with BPI-2358 (0.5 μM) and MN1-GFP⁺ murine AML cells. Treated macrophages exhibited an increased phagocytic capacity compared to untreated controls. A cell proliferation assay was performed by co-culturing BM-derived M2 murine macrophages, pre-treated with BPI-2358 (0.5 μM), with MN1-GFP⁺ murine AML cells at a 1:1 ratio. Cell growth was monitored over a 10-day period by performing regular cell counts. Notably, a significant decrease in MN1 cell proliferation was observed on days 3 and 4 in co-cultures with BPI-2358-treated macrophages, indicating that the treatment impaired leukemic cell expansion. Moreover, exposure to an AML-conditioned microenvironment induced a tolerogenic macrophage profile in vitro, which was partially reversed by treatment, highlighting the functional plasticity of these cells. Transcriptomic analysis by RNA-seq revealed differential gene expression and pathway enrichment consistent with macrophage reprogramming. Transcriptomic analysis showed that BPI-2358 upregulated 4,829 genes, including those involved in tissue damage response, tissue remodelling, and the pro-inflammatory cytokine Il1b. The treatment also significantly suppressed the IL-6/JAK/STAT3 pathway, a key driver of the immunosuppressive M2 phenotype and tumor immune evasion. Ex vivo cytotoxicity was assessed in a panel of primary AML samples (n=25) treated with BPI-2358 (0.125–0.5 µM) for 72 hours. Flow cytometry was used to evaluate viability of leukemic blasts (CD34⁺ or CD117⁺ for CD34⁻ AMLs) and macrophages (CD34⁻ or CD117⁻ HLA-DR⁺ CD14⁺) using Annexin V/DAPI staining and TMRE for mitochondrial membrane potential. Finally, the in vivo efficacy of BPI-2358 was assessed in an MN1-driven AML mouse model (MN1-GFP⁺). Mice received BPI-2358 (7.5 mg/kg IP, daily), and flow cytometry analysis of the BM showed a reduction in leukemic engraftment. The treatment did not impact the animals' body weight, suggesting minimal systemic toxicity. A significant decrease in the frequency of circulating MN1⁺ cells was observed, along with a marked reduction in MN1⁺ cell infiltration in the spleen. This was further supported by a significant decrease in spleen weight in the BPI-2358-treated group. These findings reinforce the potential of macrophage reprogramming as a complementary strategy in AML treatment, especially in molecularly high-risk subtypes such as those associated with MN1 overexpression.