Prec-O1-04 - CXCR4/CXCL12 axis as a potential therapeutic target in mycosis fungoides: an in-vitro study

Abstract

The C-X-C chemokine receptor 4 (CXCR4) is overexpressed in the tumor cells of many solid and hematologic neoplasms. Its ligand, C-X-C chemokine ligand 12 (CXCL12), is secreted by cells in the tumor microenvironment, mostly by stromal fibroblasts. The engagement of CXCL12 to CXCR4 results in activation of several intracellular signal transduction pathways, including, the proapoptotic p38, the prosurvival AKT, and the chemotaxis mediator ERK. The balance between activation of AKT and activation of p38 will determine the fate of cell viability. Plerixafor, AMD3100 (AMD), is a CXCR4 antagonist that reversibly blocks the binding of CXCR4 to CXCL12. AMD is already in clinical use as bone marrow stem cell or progenitor cell mobilizers. AMD and other CXCR4 inhibitors are investigated either alone or in combination with other systemic treatments in numerous clinical trials in cancer. CXCR4 and CXCL12 are known to be overexpressed in MF skin biopsies, but the exact differential contribution of the lymphoma vs the reactive T-cells is not clear. We previously have shown that primary MF-fibroblast culture (MF-Fs) established from MF biopsies, secrete high CXCL12 which promotes the migration of MyLa (MF cell line), protect them from chemotherapy and both effects were suppressed by AMD. We studied the role of CXCR4 as essential key protein in the survival of MF, and the effect and mechanism of CXCR4 antagonist on MyLa cell viability. We found that MyLa cells express higher CXCR4 vs CD3+CD4+ PBMCs from healthy donor (FACS). Single cell analysis of skin MF biopsy by flow mass cytometry (CyTOF) revealed sub-population of T- cells expressing the highest intensity of CXCR4, CD30, PD-1, and PDL-1 compared to all other immune cells, and therefore are suspected as the lymphoma cells overexpressing CXCR4. AMD was toxic to MyLa cells with higher toxicity to MyLa co-cultured with MF-Fs than MyLa alone (MTT viability assay, trypan blue staining), and promotes their death through apoptosis (FACS). AMD-induced death was selective to MyLa cells compared to healthy PBMCs, when both were co-cultured with MF-Fs. The phosphorylation of p38 and AKT in MyLa co-cultured with MF-Fs were reduced upon AMD treatment (Western blot). Spheroids of MyLa cells with MF-Fs were established to show the toxic effect of AMD in 3D co-culture model. The intracellular signaling pathways mediated by AMD was studied by phospho-proteomic analysis of MyLa cells co-cultured with MF-Fs. In sum, we showed the overexpression of CXCR4 in subpopulation of T cells suspected as the lymphoma cells in skin biopsy, and the pivotal role of overexpressed CXCR4 in the survival of MF cell line. The apoptotic effect mediated by AMD indicates that disrupting the interaction between stroma derived CXCL12 and MF derived CXCR4 might be a new therapeutic option in MF.