Overexpression of the ATP-binding cassette (ABC) cellular drug efflux proteins ABCB1 and ABCG2 on acute myeloid leukemia (AML) cells is associated with inferior chemotherapy outcomes. Nevertheless, inhibitors of drug transport have not improved treatment outcomes in clinical trials. The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, is expressed in AML and is implicated in regulation of multiple key cellular processes, as well as drug resistance. Our group has shown that Pim-1 phosphorylates ABCB1 and ABCG2 and promotes their translocation to the cell surface, where they mediate drug efflux. The imidazo[1,2-b]pyridazine small molecule SGI-1776 (Tolero Pharmaceuticals, Inc. Salt Lake City, UT) is the first Pim kinase inhibitor to have entered clinical testing. SGI-1776 has been shown to sensitize ABCB1-overexpressing drug-resistant cells to ABCB1 substrate cancer chemotherapy drugs, but chemosensitization was found to be associated with direct inhibition of drug transport mediated by ABCB1. Moreover, while silencing of Pim-1 expression with siRNA was found to sensitize ABCG2-overexpressing cells to ABCG2 substrate chemotherapy drugs, the effects of SGI-1776 on resistance mediated by ABCG2 have not been studied. Therefore we studied the Pim-1-dependent and -independent effects of SGI-1776 on chemosensitivity of cells overexpressing ABCB1 and ABCG2.
SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of ABCB1 and ABCG2 substrate drugs including daunorubicin and mitoxantrone 2- to 4-fold in leukemia and myeloma cell lines overexpressing ABCB1 and ABCG2, but had no effect on the IC50 of the non-substrate drug cytarabine, and no effect in parental cells. SGI-1776 also increased apoptosis of ABCB1- and ABCG2-overexpressing leukemia and myeloma cells exposed to ABCB1 and ABCG2 substrate chemotherapy drugs, respectively, and decreased their colony formation in the presence of substrate, but not non-substrate, chemotherapy drugs, with no effect on parental cells. We found that SGI-1776 decreased ABCB1 and ABCG2 surface expression, measured by flow cytometry, on K562/ABCB1 (p=0.013) and K562/ABCG2 (p=0.0038) leukemia cells, respectively, both of which express Pim-1 at high levels, without decrease in total cellular ABCB1 and ABCG2 expression, measured by Western blot analysis. In contrast, SGI-1776 had no effect on ABCB1 and ABCG2 surface expression on HL60/VCR leukemia and 8226/MR20 myeloma cells, which express ABCB1 and ABCG2, respectively, but express Pim-1 at lower levels. Thus SGI-1776 decreased ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1, consistent with decreased cell surface translocation of ABCB1 and ABCG2 as a result of inhibition of Pim-1, but also chemosensitized cells expressing ABCB1 and ABCG2 in the absence of effects on ABCB1 and ABCG2 cell surface expression. We found that SGI-1776 indeed inhibited uptake of fluorescent substrates of both ABCB1 and ABCG2, measured by flow cytometry, in a concentration-dependent manner. We further determined that SGI-1776 inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]-IAAP and stimulated ABCB1 and ABCG2 ATPase activity, consistent with binding to drug-binding sites of ABCB1 and ABCG2 and inhibition of substrate transport by both proteins. Thus SGI-1776 both inhibits drug transport by ABCB1 and ABCG2 and decreases ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1.
Pim-1 is thought to be a clinically promising therapeutic target in AML and other malignancies, and other Pim kinase inhibitors are in preclinical and clinical development. Subsequent clinically applicable Pim kinase inhibitors should be characterized with regard to interactions with ABCB1 and ABCG2. In particular, while therapeutic strategies based on inhibition of drug transport mediated by ABCB1 with competitive inhibitors including PSC-833, zosuquidar and cyclosporin A have largely been clinically unsuccessful, inhibition of ABCB1 and ABCG2 cell surface translocation by Pim kinase inhibitors may have therapeutic implications.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.