PS1305 CHARACTERIZATION OF ENDOGENOUS CXCR4 INHIBITORY PEPTIDES TO TARGET WALDENSTRÖM'S MACROGLOBULINEMIA
Authors: L.M. Kaiser, C. Buske, J. Münch, D. Sauter
Journal: HemaSphere (2019)

Background: Waldenströḿs Macroglobulinemia (WM) is an indolent B‐cell lymphoma with elevated monoclonal IgM serum levels and bone marrow infiltration of lymphoplasmacytic cells. Whole‐genome sequencing has led to identification of two prevalent mutations affecting MYD88 and CXCR4 that allow the classification into 3 genotypes. It was shown that CXCR4 mutations contribute to malignant growth and are associated with Ibrutinib resistance in WM. So far, naturally occurring mechanisms counteracting CXCR4 driven proliferation in cancer are largely unknown. We screened a human blood‐derived peptide library in order to identify novel anti‐cancer peptides that target CXCR4. This approach succeeded in identifying an as‐yet‐unknown CXCR4 blocking peptide called EPI‐X4 (endogenous peptide inhibitor of CXCR4) that is generated naturally in the human body. The 16‐mer peptide is cleaved from its precursor albumin under acidic conditions and was shown to specifically interfere in the cross‐talk between CXCR4 positive cancer cells and their environment. Successful development of highly active and more stable EPI‐X4 derivatives (WSC02 and JM#21) could be of clinical relevance in the future. Aims: Our goal is to analyse the functional role of the naturally occurring EPI‐X4 and to test optimized EPI‐X4 derivatives as potential therapeutic tool in WM. Methods: We performed multiple cell line based functional assays such as apoptosis and migration as well as experiments with EPI‐X4 treated WM cells in vitro and in vivo using NSG mice. Overexpression of CXCR4 mutations was achieved via lentivirus‐mediated infection of WM cells. Whole transcriptome analysis based on next‐generation sequencing was carried out using the Illumina platform and validated via Real‐Time PCR. Proteomic analysis was done using highly sensitive tandem mass spectrometry. Results: We have investigated the effect of EPI‐X4 and its optimized derivatives on blocking the CXCR4 receptor, WM cell migration and inhibition of cell proliferation in presence or absence of different CXCR4 mutations. Efficient and dose depending blocking of CXCR4 by the peptides was documented by two different clones of CXCR4 antibodies, recognizing different epitopes. Both optimized forms of EPI‐X4 were able to abrogate growth and efficiently induced apoptosis in WM cells (up to 90% compared to the control). Migration of WM cells with or without activating CXCR4 mutations was inhibited by 50% for the naturally occurring EPI‐X4 and 95% for the optimized peptides compared to the control. Mice injected with WSC02 treated WM cells showed a prolonged survival of 26 days (median survival control 54,5 days versus 80,5 days for WSC02, respectively). To better understand the impact of EPI‐X4 on the transcriptome, we profiled EPI‐X4 treated cells at the mRNA level using RNA‐seq technique. By performing gene enrichment analysis, genes related to carbohydrate and lipid metabolism pathways as well as the RAF/RAS pathway, hypoxia signal cascades and p53 were enriched. Proteomic changes upon EPI‐X4 treatment on different WM cells are currently being analyzed. Summary/Conclusion: Collectively, our data indicate that naturally occurring mechanisms exist that counteract CXCR4 induced lymphoma growth and that this can be used to develop innovative treatment concepts which target CXCR4 mutations in WM.