Anti-Leukemic Activity Is Increased with Low Dose Busulfan and Irradiation: A Preclinical Model

Abstract 4696

Combinations of busulfan (Bu) and low dose total body irradiation are commonly used in conditioning regimens for allogeneic stem cell transplant in acute myeloid leukemia (AML). In this study we tested the in vitro effect of physiologic doses of Bu and ionizing radiation (IR) to address the question whether additive or synergistic antileukemic activity can be demonstrated in two leukemic cell lines, one sensitive (HL60) and one resistant (K562) to radiation.

Cells were treated for 24 hours with Bu at 0, 12.5, 25, 50, 100 or 200 μ g/ml, or IR at 1.5, 3 or 6 Gy, or a combination of both with 12.5 or 25 μ g/ml for 24 hours, followed by IR at 1.5 or 3Gy. Cells were then tested for proliferation by thymidine uptake, apoptosis by surface expression of annexin V and caspase 3, and in-vitro colony formation assays. Exposure to Bu or IR for 24, 48 and 72 hours induced a dose dependent inhibition of proliferation and colony formation in both cell lines. However, 3 or 6 Gy of IR were both very effective against HL60 cells whereas a significant increase in cytotoxicity in K562 cells was observed only by using 6Gy (p=0.008).

In-vitro treatment with either Bu or IR caused apoptotic cell death mainly in HL60 cells, but in a very limited amount in K562 cells. To test the effect of the combination of Bu and IR we utilized a low dose of Bu for 24 hours with low doses of radiation. HL60 cells were strongly inhibited by both agents when separate or combined. In contrast, treatment of the relatively radio-resistant K562 cells with low dose IR (3Gy) alone inhibited colony formation by 28%. The inhibitory effect increased to 84% when combined with low dose Bu.

In order to identify genes associated with a response to Bu, we formed a linear regression model controlled for cancer type and also leukemic cell lines using GI₅₀ and Stanford cDNA array data from the NCI-60. Among 7 genes (p<0.001 and FDR ∼50%) that were identified for all cancers, CLK-2 was also shown to correlate with response to Bu in leukemic cell lines (p=0.047). We then tested how these significant genes performed in the Affymetric U133 plus 2.0 platform. Six other genes (ERC2, HCLS1, CD74, KCNH2, HLADQB2, CD53) were significantly associated with response to Bu (p<0.05).

In conclusion, we demonstrated an additive effect of Bu and low dose IR even on radioresistant leukemic cells. These findings prompted the design of a clinical trial with IV Bu and marrow irradiation in the conditioning regimen prior to allogeneic stem cell transplant for acute leukemia. Expression of genes correlating with response to Bu may represent a novel biomarker to predict the outcome of leukemic patients undergoing stem cell transplant and could lead to a personalized anti-leukemic therapy with Bu.