HGF Regulation in the Tumor-Supportive Microenvironment Combined with Pan-PIM Kinases Inhibition in Malignant Cells As a Multi-Target Approach for Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), persistent clonal proliferation of malignant myeloid cells results in high severity and low cure potential. Resistance to currently available chemotherapeutics and disease relapse remains an important concern worldwide. Several molecular targets play key roles in impairing therapeutic response, including PIM-kinases, a serine/threonine kinase family associated with the progression of hepatocyte growth factor (HGF)-driven cancers through its receptor, MET. We reported that enhanced HGF secretion by bone marrow (BM) mesenchymal stromal cells (MSC) correlated with low levels of the serine protease inhibitor kunitz‐type2 (SPINT2) in AML (Roversi et al., 2019), which are affected by immunosuppressive properties of the tumor microenvironment (TME). However, the interplay between SPINT2, HGF, and PIM-kinases in the TME is yet to be elucidated and could represent an important multi-target therapeutic strategy in hematological cancers.

Aims: Given the importance of HGF to AML prognosis and progression, we analyzed the interplay between SPINT2, HGF, and pan-PIM kinases by evaluating the gene expression and the effects of a novel pan-PIM kinase inhibitor, PIM447 (Novartis), in leukemia cells and in the TME.

Methods:SPINT2, HGF, PIM1, PIM2 and PIM3 mRNA expression data were obtained from OHSU (2018) AML study and TCGA (cBio Portal). Correlation analysis was obtained by Spearman's coefficient (r). A total of 5 healthy donors (HD) and 5 de novo AML patients BMMSC (CD90+CD105+CD73+CD45-CD34-CD31-HLA-DR-) were treated with 5-Azacitidine (Aza), and evaluated for SPINT2 gene expressions. Myeloid (OCI-AML3, MOLM13, U937) and lymphoblastic (RS4;11, Jurkat) leukemia cells were treated with increasing concentrations of PIM447, and the 101nd apoptosis rates were determined. Peripheral blood and BM mononuclear cells (PBMC/BMMC) from HD were tested for cytotoxicity. Leukemia cells were also examined for reactive oxygen species (ROS), nitric oxide (NO) production, and mitochondrial membrane potential (ΔΨm). To verify PIM447 action onto an immunosuppressive TME, cocultures of leukemia cells and monocytes or monocyte-derived macrophages (MDMs) were performed. To understand the interplay between SPINT2 and PIM-kinases, cocultures of SPINT2-overexpressed MSC and leukemia cells, were performed. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests ( P<.05).

Results: The analysis of gene expression performed in MC from 188 AML patients (age median: 57.5 years [range: 18-88]) showed that levels of HGF correlated positively with PIM1, PIM2, and PIM3 ((r) =.8727;.9094;.9093, respectively, P<.0001) and negatively with SPINT2 (r=-.06402). We also verified that SPINT2 mRNA expression was upregulated in AML-BMMSC ( P<.05) after Aza treatment when compared to HD-BMMSC, suggesting that this gene is methylated in AML. Our in vitro results showed PIM447 treatment (2-20μM) yielded reduced cell viability and increased apoptosis in leukemia cells [IC50 range: 12.2-22.2μM, P<.01] without affecting HD cells. PIM447 reduced ROS, NO production, and ΔΨm (all P<.0001) in all cell lines. PIM447 treatment of macrophages differentiated from HD-PBMC revealed a shorter population of M2 macrophages (HLA-DR-CD206+) ( P<.001) and an increased population of M1 macrophages (HLA-DR+CD80+) ( P<.05). Moreover, PIM447 promoted apoptosis of leukemia cells co-cultured with MDMs ( P<.0001). Interestingly, co-culture analyses of SPINT2-overexpressed MSC enhanced PIM447 cytotoxic activity towards leukemia cells (P<.05) and reduced cell-cell adhesion by diminishing CD49b, CD49d, and CD49e (P<.05), suggesting an interplay between SPINT2 and pan-PIM kinases

Conclusion: Collectively, our data identify a novel pan-PIM kinase inhibitor, PIM447, which effectively induces leukemia cell apoptosis through reducing MET receptor activation as well as acts on their tumor-supportive microenvironment by inducing a phenotypic shift from immunosuppressive M2 to anti-tumor M1 macrophages. PIM447 also interplays with SPINT2/HGF pathway in the TME, reducing AML progression. These findings suggest that a pivotal strategy to treat AML could be a multi-target therapy, acting both on leukemia cells and in the tumor microenvironment.

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