The Manually Designed CXCR4 Targeting Peptide Is Used In Basic Research On AML Therapy

Objective:According to the latest American cancer statistics, large numbers of new acute myelocytic leukemia (AML) cases and deaths are emerging every year. Poor prognosis of AML is mainly due to the infiltration and chemoresistance of AML cells, minimal residual disease and relapse. Bone marrow stroma can recruit and protect AML cells against chemotherapeutic agents, and provide anti-apoptosis signals and favorable conditions for survival and growth through secreting stromaderived factor 1α (SDF-1, CXCL12) to activate its receptor CXCR4 highly expressed on AML cells, which results in minimal residual disease and relapse. Thus disrupting the CXCR4/CXCL12 axis with antagonists is of significance for improving the chemosensitivity and decreasing relapse rate of AML. Due to the advantages of designed peptides, such as low cost, low immunogenicity, synthesis flexibility, we designed novel peptides according to the sequence feature of CXCR4 and from which we screened one peptide that can inhibit CXCR4/CXCL12 axis-induced AML cell migration and adhesion. We combined this peptide with different chemotherapeutics and determined its effect in vitro (AML cells) and in vivo (AML mice model). Meanwhile we evaluated the safety of peptide in healthy mice.Methods:The affinity of peptide to multiple human AML cells (HL-60, NB4, THP-1, U937) expressing CXCR4 was examined using flow cytometry. The effect of peptide on the apoptosis of AML cells and nonmalignant cells (murine stromal cell MS-5 and human umbilical vein cell ea.hy926) was tested by Annexin V/PI double staining and caspase-3 activity assay. By transwell assay and adhesion assay, the effect of peptide on CXCR4/CXCL12-induced AML cell migration and adhesion was determined. By confocal microscopy analyses and western blot, the effect of peptide on CXCL12-induced actin microfilament reorganization and the phosphorylation of Erk, Akt and p38 was examined. By Annexin V/PI double staining and trypan blue exclusion method, the combination effect of peptide and chemotherapeutics on apoptosis and viability of AML cells co-cultured with MS-5 was examined. The peptide-induced AML cells mobilization was detected with CD33 using flow cytometry in AML mice. The combination therapy effect of peptide and chemotherapeutics on AML mice was evaluated by the AML mice survival, the leukemia burden of bone marrow, spleen and peripheral blood using flow cytometry, and the AML cell infiltration into the spleen and liver using histological analysis. The safety of peptide was evaluated in healthy mice using the histological analysis of tissue sections and routine clinical parameters of serum analysis.Results:(1) Screened peptide (named E5) has high and stable affinity to multiple AML cells with high CXCR4 level. (2) E5 significantly inhibits CXCL12- or murine stromal cell (MS-5)-induced migration of AML cells and prevents the AML cells from adhering to MS-5 cells in a concentration dependent manner. (3) E5 with concentration higher than 20 μM can induce apoptosis in the four AML cell lines tested while does not affect the viability of nonmalignant cells MS-5 or human umbilical vein cell (ea.hy926), both of which have a low level of CXCR4. (4) Mechanistic studies demonstrate that E5 down-regulates CXCL12-induced phosphorylation of Akt, Erk, and p38, which affects the cytoskeleton F-actin reorganization and ultimately results in the inhibition of CXCL12- and stroma-mediated leukemia cell responses. (5) Addition of E5 to the co-culture system of AML cells and MS-5 can significantly increase the AML cell apoptosis induced by vincristine, cisplatin and 10-hydroxy camptothecin after 48 h, and induce significant reduction of AML cell viability compared with a constant high viability level in the mono-chemotherapy group after 10 days. E5 can reverse the protection of MS-5. (6) In an AML mousexenograft model, E5 induces 1.84-fold increase of circulating AML cells out of protective stroma niche. (7) Combined with cyclophosphamide or vincristine, E5 decreases the leukemia burden of bone marrow, spleen and peripheral blood, inhibits infiltration of AML cells into spleen and liver, and prolongs the lifespan of AML mice compared with mice treated with chemotherapy alone. (8) No necrosis or inflammation is observed in the H&E staining tissue section of E5-treated healthy mice, and the hepatic and renal function of mice are normal according to routine clinical parameters of serum analysis. No organ weight change is observed in E5-treated mice.Conclusion:Through inhibiting CXCR4/CXCL12 axis-mediated migration and adhesion of AML cells to bone marrow stroma, the novel synthetic peptide E5 inhibits the infiltration of AML cells, mobilizes AML cells out of the protective bone marrow microenvironment and enhances the therapeutic efficiency of various chemotherapeutics on AML in vitro and in vivo. E5 has good safety. Significant benefit of E5 can be expected in the leukemia therapy in combination with chemotherapies.