Inhibition of MET Tyrosine Kinase Provides a Strategy for Targeting the Inflammatory Tumor Microenvironment

Background: In acute myeloid leukemia (AML), age-associated inflammation may contribute to leukemia progression and remodel the tumor microenvironment (TME). Macrophages, crucial components of the TME, play a critical role in modulating T cell immune response against leukemia cells. However, macrophage polarization to an M2 phenotype, known as tumor-associated macrophages (TAMs), have an immunosuppressive action and interplay with leukemia cells by promoting their survival, immune evasion, and chemotherapy resistance. MET, a hepatocyte growth factor (HGF) receptor, has been demonstrated to contribute to shifting macrophages to TAMs. Hence, we hypothesize that inhibiting Met and, consequently, HGF action and signaling could be a potential strategy to target the inflammatory TME and improve AML clinical outcomes.

Aims: Here,we analyze the MET gene expression in leukemia patients mononuclear cells and the impact on clinical outcomes and we also assess the contribution of a specific MET inhibitor, B2000 (Brigham University), to leukemia progression by investigating the in vitro, ex vivo and in vivo effects with focus on the inflammatory TME.

Method s:MET mRNA expression data were obtained from TCGA AML study, performed in cells isolated from 290 AML patients (median age 61 years [range:18-87]). Human peripheral blood and bone marrow (BM) mononuclear cells (BMNC/PBMC) (n=10) were obtained from healthy donors (HD). HD-PBMC were used for the isolation of monocytes. Human leukemia cell lines (OCI-AML3, MOLM-13) and BMNC from HD and AML patients (n=10) were treated with B2000 and apoptosis rates were evaluated. To obtain monocyte-derived macrophages (MDMs), monocytes were stimulated with GM-CSF/LPS/IFN-y or M-CSF/IL-4, for M1 or M2 polarization, respectively, and treated with B2000 in monocultures and cocultures. For in viv o analyses, BALB/c mice received a single intraperitoneal injection of lipopolysaccharide (LPS) to provoke an inflammatory shock and were treated with B2000. The analysis of the immune response and the characterization of inflammatory cells was performed by flow cytometry.

Results: High MET expression in FAB M1, M3 and M7 AML patients ( P<.0001) was observed. B2000 was effective in killing leukemia cell lines ( P<.0001) and primary BMNCs from AML patients ( P<.005) without affecting HD-BMNC. HD-PBMC-derived monocytes treated with B2000 had increased classical monocyte population (CD14highCD16-) ( P<.001). B2000 also caused a significant apoptosis increase in leukemia cells cocultured with monocytes, without modulating the monocytes population ( P<.0001). To confirm whether monocytes could acquire a tumoricidal phenotype, monocytes were pre-treated with B2000 and, then, cocultured with leukemia cells. B2000-programmed monocytes enhanced leukemia cell apoptosis ( P<.001). The analysis of MDMs revealed a shorter population of M2 MDMs (HLA-DR-CD206+) and increased population of M1 MDMs (HLA-DR+CD80+) in B2000-treated MDMs when compared with the vehicle treatment ( P<.0001). To validate these findings, we evaluated B2000 action in the inflammatory TME by co-culturing leukemia cells with MDMs and results showed that apoptosis was increased in leukemia cells in the presence of B2000 compound ( P<.01). The in vivo analysis in an inflammatory mice model showed increased frequency of M1 monocytes (CD11b+F4/80+CD80+) and decreased M2 (CD11b+F4/80+CD206+) in B2000-treated ( P<.05). Accordingly, B2000 restored lymphocyte B ( P<.05) and T ( P<.05) levels in the BM, spleen and peripheral blood, decreasing the inflammatory burden.

Conclusion: We demonstrate the pre-clinical efficacy of a MET inhibitor, B2000, in enhancing anti-tumor immunity in vitro, ex vivo and in vivo by promoting M1 macrophage polarization and tumoricidal phenotype, contributing to inhibition of leukemia cell expansion and inducing the specific leukemia apoptosis. B2000 also acted on restoring lymphocyte levels and, consequently, reducing inflammatory stress. Our findings highlight an important role of this novel MET-inhibitor onto the inflammatory TME, representing an important agent against hematological neoplasms with a novel immunomodulatory effect in directly reducing leukemia cell proliferation as well as promoting TAMs anti-tumoral effector functions.

Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.