MerTK Receptor Tyrosine Kinase Inhibition As a Potential Strategy to Augment Immune-Mediated Clearance of Acute Myeloid Leukemia

The MerTK receptor tyrosine kinase is aberrantly expressed in 80-100% of acute myeloid leukemia (AML), and inhibition of MerTK prolongs leukemia-free survival in vivo, making it an appealing therapeutic target. MerTK is normally expressed on macrophages (MMs) and dendritic cells (DCs) and facilitates engulfment of apoptotic debris. Through this process, MerTK promotes tolerogenicity and anti-inflammatory cytokine production. We hypothesized that inhibition of MerTK will also mediate anti-leukemia effects through relief of microenvironment immunosuppression and stimulation of innate MM and DC activity leading to enhanced T-cell immunity.

To test this hypothesis, we transplanted wild-type C57BL/6 mice with syngeneic murine MLL-ENL fusion AML or primary mouse MLL-AF9 AML. We evaluated the effects of MerTK inhibition using MRX-2843, a MerTK kinase inhibitor. Mice were treated daily and survival was monitored. Treatment with MRX-2843 prolonged median survival (43 days) compared to vehicle treatment (17 days) in mice transplanted with MLL-ENL AML (p<0.0001), and MLL-AF9 primary murine AML (42 days with MRX-2843, 30 days with vehicle; p<0.05).

To explore the immune effects of MerTK inhibition in AML, we evaluated in vivoMMs and DCs by flow cytometry. Compared to non-leukemic controls, vehicle-treated leukemic mice demonstrated a significant infiltration of MMs and DCs into the primary sites of leukemic burden (spleen, bone marrow). These MMs and DCs expressed high levels of co-inhibitory ligands PD-L1 and PD-L2. Treatment with MRX-2843 significantly decreased PD-L1 and PD-L2 expressing MM and DCs in the spleen (p<0.01) and bone marrow (p<0.01) down to non-leukemic levels. Similar results were not obtained when mice were treated with the non-MerTK targeted FLT3 inhibitor AC220, suggesting that these effects are likely specific to MerTK inhibition.

We next evaluated effects of MerTK inhibition on the leukemia microenvironment. We confirmed that T-cells do not express MerTK in-vivowith or without AML, nor +/- MRX-2843. Compared to non-leukemic mice, leukemic mice significantly upregulated co-inhibitory receptors PD-1 and Tim-3 on CD4 and CD-8 T-cells in the spleen and bone marrow. Treatment with MRX-2843 in leukemic mice significantly decreased CD4 and CD8 T-cell PD-1 and Tim-3 expression in the spleen (p<0.05) and bone marrow (p<0.05) down to non-leukemic levels. We next evaluated the role of functional T-cells in the anti-leukemia effects of MerTK inhibition. Wild-type mice and mice harboring a T-cell receptor mutation (TCRa-/-) were transplanted with MLL-ENL AML, treated with MRX-2843 or vehicle, and monitored for symptoms of leukemia. Median survival of all vehicle-treated mice with leukemia was 20-22 days. Median survival of TCRa-/- mice treated with MRX-2843 was 30 days, whereas median survival of MRX-2843 treated wild-type mice was significantly extended to 40 days (p<0.01). To confirm these findings, leukemic wild-type mice were CD8 depleted and treated as above. Median survival of CD8-depleted MRX-2843 treated mice was significantly diminished (30 days) compared to isotype (control) + MRX-2843 treatment (40 days, p<0.01). These data suggest an additional non-cell autonomous role of T-cell anti-tumor immunity in mice treated with a MerTK inhibitor.

We next evaluated peripheral serum cytokines using Luminex magnetic bead analysis. Treatment with MerTK inhibition significantly increased serum [IL-18] (p<0.01) and [MCP-3/CCL7] (p<0.05) by 2-fold compared to vehicle-treated mice. Additionally, there was no correlation between co-inhibitor expression and serum [IFN-g]. These changes appeared to be mediated through MerTK and not solely attributable to tumor burden given that MLL-ENL cells in culture did not exhibit these changes +/-MRX-2843. However, MLL-ENL cells did secrete significantly less IL-4 (p<0.01) and LIF (p<0.05) with MerTK inhibition in vitrocompared to vehicle treatment.

In conclusion, inhibition of MerTK decreased infiltration of immunosuppressive co-inhibitory ligand-expressing MMs and DCs into primary sites of leukemic engraftment. Furthermore, inhibition of MerTK altered the leukemia microenvironment to decrease T-cell co-inhibitory receptor expression and improve anti-leukemia effects. These preliminary studies demonstrate the potential of MerTK inhibition as an immunotherapeutic strategy in AML.