Synergistic Killing of E2A-PBX1+ B-ALL Cells by Chemotherapeutic Agents and MerTK Inhibition
Authors: Linger Rachel M.A. , DeRyckere Deborah , Brandão Luis , Sawczyn Kelly K. , Keating Amy K. , Graham Douglas K. 
Published: April 2020

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although cure rates have increased in recent years, significant risk of both short- and long-term toxicities persists including a 25% incidence of a severe late effect in pediatric cancer survivors. Thus, novel and less toxic therapies are needed. Most pediatric leukemias are of the B-cell lineage and a subset of these express the fusion protein E2A-PBX1 as a result of the chromosomal rearrangement t(1;19). A previous DNA microarray study suggested that the receptor tyrosine kinase Mer (MerTK) is aberrantly expressed in E2A-PBX1+ B-ALL (Yeoh et al., 2002, Cancer Cell 1:133–143). Within the hematopoietic lineages, MerTK is expressed in dendritic cells, monocytes/macrophages, NK cells, NKT cells, megakaryocytes, and platelets. However, MerTK is not expressed in normal lymphocytes. In studies of T-cell ALL, we have previously shown that ectopic expression of MerTK contributes to leukemogenesis. MerTK is known to activate anti-apoptotic signaling proteins such as Akt and Erk 1/2. These data led us to hypothesize that abnormal expression and activation of MerTK may provide a survival advantage for leukemia cells. Furthermore, inhibition of MerTK may enhance the sensitivity of leukemia cells to cytotoxic agents. In this study, we extended the findings of Yeoh et al. (2002) by confirming the ectopic expression of MerTK mRNA and protein in patient samples and human cell lines of E2A-PBX1+ B-ALL. In subsequent studies, we used lentiviral short hairpin RNA (shRNA) contructs to knockdown MerTK in the human E2A-PBX1+ B-ALL cell line, 697. In vitro assays of cell proliferation and survival demonstrated that inhibition of MerTK significantly increased the sensitivity of 697 cells to numerous chemotherapeutic agents. For example, when wildtype or non-silencing shRNA control (shControl) 697 cells were treated with 6-mercaptopurine (6-MP), an IC50 could not be determined because the cells were robustly resistant to killing with this agent. However, the MerTK knockdown lines demonstrated remarkable sensitivity (IC50 values ~ 4 μM) to 6-MP. In other cases, the wildtype/shControl 697 lines were sensitive to treatment with chemotherapeutic agents but a dramatic increase in sensitivity was observed with MerTK inhibition. These results were acquired via an assay of metabolically active cells which precludes determination of the contribution of changes in cell proliferation and cell death. To more specifically evaluate the role of cell death, we performed flow cytometric analysis of cells stained with propidium iodide and YO-PRO®-1. These experiments revealed that inhibition of MerTK results in increased induction of cell death in response to treatment with chemotherapeutic agents. Western blotting was used to compare the signaling pathways activated in shControl versus MerTK knockdown 697 cells following treatment with chemotherapeutic agents. Inhibition of MerTK leads to loss of ERK 1/2 and mTOR signaling in 697 cells. Further investigation demonstrated that inhibition of MerTK also correlates with robust activation of apoptotic proteins in response to treatment with chemotherapeutic agents. Specifically, cleavage of both Caspase 8 and Caspase 9 was observed suggesting that inhibition of MerTK permits activation of both intrinsic and extrinsic apoptosis pathways in response to treatment with chemotherapeutic agents. Taken together, these in vitro results suggest that inhibition of MerTK may increase the efficacy of standard chemotherapy and thereby allow for dose reduction and decreased toxicity. To demonstrate proof of concept in an in vivo setting, we evaluated the oncogenicity of wildtype, shControl and MerTK knockdown 697 cells in a mouse xenograft model of leukemia. We found that MerTK inhibition significantly delayed the onset of disease. Animals which received wildtype or shControl 697 cells exhibited median survival of 22 and 26 days, respectively. Median survival was significantly increased to 63 days for each of two clonal MerTK knockdown lines (P&lt;0.0001). Our results demonstrate that MerTK is a novel biological target for treatment of leukemia and possibly a spectrum of other cancers known to aberrantly express MerTK.