Src Kinase Inhibitors Enhance ATRA-Mediated Differentiation of Myeloid Cells.

Myeloid leukemias are characterized by a blockade in differentiation resulting in accumulation of proliferating progenitor cells and a lack of mature granulocytes and monocytes. In acute promyelocytic leukemia (APL), a subtype of AML, treatment with all-trans retinoic acid (ATRA) has been shown to overcome this blockade in differentiation and is the current standard of care for APL patients. However, other forms of myeloid leukemia show no response to ATRA. Src family kinases (SFK) have been shown to be activated during normal myeloid development by both proliferation-inducing cytokines, such as IL-3, and differentiation-promoting factors, such as G-CSF. To elucidate the role of src family kinases during myeloid differentiation, we examined the impact of SFK inhibitors, PP1 and PP2 on ATRA-mediated differentiation of myeloid cell lines. Interestingly, PP1 potentiated ATRA-mediated myeloid differentiation of HL-60 cells, inducing a two-fold increase in differentiated cells at 72 hours as determined by CD11b staining, NBT reduction and morphological analysis. To determine whether enhancement of ATRA-mediated differentiation was specific for PP1, or could be promoted by a different src family kinase inhibitor, we evaluated the impact of PP2. PP2 was found to exhibit similar effects on ATRA-induced myeloid differentiation of HL-60 cells. Additionally, both PP1 and PP2 enhanced ATRA-induced monocytic differentiation of U937 cells and granulocytic differentiation of NB-4 cells. Interestingly, PP1 was found to enhance ATRA-induced differentiation even when added 24 hours after addition of ATRA. The impact of PP1 on ATRA-mediated myeloid differentiation was dependent on signaling via the MEK/ERK pathway since pre-treatment with U0126, a MEK-1/-2 inhibitor, attenuated myeloid differentiation induced by the combination of RA and PP1. Reporter assays utilizing an RARE-luciferase construct indicate that PP1 and PP2 do not enhance ATRA-mediated differentiation by promoting the transcriptional activity of the retinoic acid receptor (RAR). Together, our results demonstrate that PP1 and PP2 potently enhance ATRA-induced myeloid differentiation of AML cell lines and suggest that src inhibition in combination with ATRA may be a useful treatment for AML. Studies evaluating the impact of src inhibition on ATRA-mediated differentiation of primary AML cells are currently underway.