Session: 615. Acute Myeloid Leukemia: Therapy, excluding Transplantation: Poster III
Methods: In the present study, the effect of the high affinity CXCR4 antagonist BL-8040 (BKT140) and AMD3100 (Mozobil) alone and in combination with ARA-C or the FLT-3 inhibitor AC220 on the survival and proliferation of AML cells in-vitro was examined. In the in-vitro study HL60 (FLT3-WT), MV4-11 (FLT3-ITD) cell lines and human primary AML cells from patients with FLT3-ITD mutations and FLT3-WT genewere used. Cells were incubated for 48 hrs in the presence of BL-8040 (8µM-20µM), AMD3100 (20µM), ARA-C (10-200 ng/ml) and AC220 (0.5-50nM). The level of viable cells, percentage of apoptosis and cell cycle were evaluated by FACS using propidium iodide and 7-AAD. In-vivo, we used NOD scid gamma (NSG) mice engrafted with human primary AML blasts and explored the effects of single injection of BL-8040 on the mobilization and survival of the blasts in the blood and the BM of the engrafted mice.
Results: In-vitro, treatment of MV4-11 cells (FLT3-ITD) with BL-8040, unlike treatment with AMD3100, directly inhibited cell growth by 35% and increased cell death by 39%. Furthermore, in-vitro, treatment of primary AML cells (FLT3-ITD) with BL-8040 directly inhibited cell growth by 28-47% and increased cell death by 75-100%. A combination of BL-8040 with AC220 or ARA-C further increased the apoptotic effect of these agents achieving a 96% reduction in cell viability and inducing cell death by 70- 90% of AML cells. When we studied the in-vitro effect of these agents on FLT3-WT cells (HL-60 cell line and primary AML cells), we found that BL-8040 inhibits cell growth by 16-50%. Unlike the FLT3-ITD cells, in the FLT3-WTcells we did not observe additive effects on cell growth for the combined treatments of BL-8040 with AC220. The combined treatment of BL-8040 with ARA-C was found to further increase the percentage of AML cell death. Moreover, BL-8040 decreases the percent of cycling cells by reducing the number of cells in G2/M+S phase while increasing the number of apoptotic cells in sub-G0 phase. It is interesting to mention that the migration of all tested AML cells toward CXCL12 was entirely inhibited by BL-8040. In-vivo, we found that a single injection of BL-8040 (100μg/mice) into NSG mice engrafted with human AML cells, induces rapid mobilization of AML cells to the periphery within 4 hrs after injection (an 8 fold increase from the control). When mice were administered with 5 consecutive injections of BL-8040 (400μg/mice), a reduction in the number of AML blasts in the blood was observed (40-60% reduction) with induction of AML cell apoptosis within the BM and in the blood by 10-20-fold, compared to the control.
Conclusions: The CXCR4 antagonist BL-8040 was found to rapidly and efficiently induces cell death of AML cells both in-vitro and in-vivo. These results suggest potential therapeutic advantages of BL-8040 in both FLT3-positive and negative AML patients by targeting not only AML anchorage in the BM but AML survival as well. Furthermore, it could provide a rational basis for BL-8040 therapy in combination with ARA-C and the FLT3 inhibitor AC220.
Disclosures: Eizenberg: Biokine: Employment . Pereg: BioLineRx LTD: Employment . Klapper: BioLineRx LTD: Employment . Abraham: Biokine: Employment .
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