CXCR4 Signaling in AML Is Dampened By the Endogenous CXCR4 Inhibitory Peptide EPI-X4

CXCR4 – CXCL12 interaction belongs to one the most fundamental loops regulating in particular cell anchorage in the BM and trafficking of cells to distant organs. This regulatory principle is highly conserved between species and is also maintained in neoplasms, shaping the behavior of malignant cells. CXCR4 is overexpressed in 25–30% of patients with acute myeloid leukemia (AML) and is associated with poor prognosis. Functional assays have proven that the CXCR4 – CXCL12 crosstalk is critical in regulating the interaction between leukemic stem cells (LSCs) and their niche. We now demonstrate that an endogenous peptide called EPI-X4 is a key candidate to balance CXCR4 signaling in AML. EPI-X4 was identified as a peptide (EPI-X4 endogenous inhibitor of CXCR4) specifically binding to CXCR4. It is generated at low pH chopped from serum albumin by Cathepsin D and E, forming a 16-mer peptide. By screening 283 primary AML patient samples, we could demonstrate that AML bulk as well as functionally validated human AML-LSCs are highly positive for CXCR4. Upon EPI-X4 treatment we could observe a marked deprivation in clonogenic growth and migration of CXCR4 positive AML cells towards a CXCL12 gradient (reduction by 66 and 77 % compared to inactive peptide as control, respectively; p<0.0005 and p<0.03). The growth inhibitory effect of EPI-X4 was accompanied by a significant decrease in CXCL12 stimulated phosphor-ERK levels, suggesting reshuffling of signaling pathways by the peptide. Furthermore, homing and engraftment potential of primary CXCR4 expressing AML patient sample in NSG mice was reduced by EPI-X4 (engraftment by 76%; p<0.004). These antagonizing and intrinsic effects were even increased when treating the cells with optimized derivatives of EPI-X4 with higher anti-CXCR4 activity. We further performed RNA-Seq on the AML cell line OCI-AML3 treated with EPI-X4 or an inactive control for 24 h in serum free conditions. In total, we found 629 genes differentially expressed between EPI-X4 and the control (FDR<0,05; Fold change >0,5). Detailed gene enrichment analysis revealed alteration of genes involved in carbohydrate and lipid metabolism pathways. The same set-up was used to assess the protein profile of EPI-X4 treated OCI-AML3 cells and resulted in quantification of 3500 proteins of which 26 were differentially expressed, among them PYCARD involved in Caspase – mediated apoptosis regulation and ECD, associated with TP53 stability. Taken together, these data demonstrate that a naturally occurring peptide exist, which counteracts CXCR4 signaling in AML. Understanding these endogenous systems which balance CXCR4 activity in malignant diseases will finally help to develop novel therapeutic tools by generating optimized highly specific anti-CXCR4 peptides.