Inhibition of the Serine/Threonine Kinase Pim-1 Has Anti-Proliferative Effects In Acute Myeloid Leukemia (AML) and Sensitizes Multidrug Resistant Cells to Chemotherapy

Abstract 1832

Poster Board I-812

The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, phosphorylates and thereby increases expression of multiple cellular proteins, including the pro-apoptotic protein BAD, the cell cycle regulatory proteins p21, p27, Cdc25A and Cdc25C, the transcription factors SOCS-1, RUNX3 and c-myc and, as we recently demonstrated, the drug resistance-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Pim-1 is synthesized in an active form by virtue of its hinge structure, and its activity is therefore regulated solely by its level of expression. Pim-1 is overexpressed downstream of FLT3 in AML cells with FLT3-ITD, but less is known about its expression and role in AML with wild-type (wt) FLT3. We studied Pim-1 expression and the effects of Pim-1 inhibition on AML cell survival, proliferation, apoptosis and chemosensitivity. Cell lines studied included HL60, K562, U937, Kasumi-1 and EOL-1 FLT3-wt cells and MV4-11 and MOLM-14 FLT3-ITD cells, as well as Pgp+ HL60/VCR and BCRP+ 8226/MR20 and parental 8226 myeloma cells as a model for BCRP-mediated drug resistance. Expression of Pim-1 and of phospho-BAD at S112, a measure of Pim-1 activity, was studied by Western blot analysis, normalized to GAPDH expression. Effects of the Pim-1 inhibitor SGI-1776 (SuperGen, Inc., Dublin, CA) on survival, cell cycle, apoptosis and colony growth were measured in WST-1 cell survival, flow cytometric cell cycle and apoptosis, and methylcellulose colony formation assays, respectively. SGI-1776 inhibits Pim-1 at a concentration of 7 +/− 1.8 nM, but is more than 90% bound to human plasma protein, so that its Pim-1 inhibitory concentration in cell culture-based assays is in μM range. Of note, SGI-1776 also inhibits FLT3 in this concentration range. Pim-1 was expressed in all cell lines studied, and expression of Pim-1 and of phopho-BAD did not differ between FLT3-ITD and FLT3-wt cells, nor between drug-resistant and parental cells. SGI-1776 decreased viable cell numbers in 96-hour WST-1 cell viability assays, with IC₅₀'s of 5 to 7 μM in FLT-wt cells, while IC₅₀'s were 20 and 65 nM, respectively, in MV4-11 and MOLM-14 FLT3-ITD cells. SGI-1776 IC₅₀'s did not differ between Pgp+ or BCRP+ cells and parental cells. In FLT3-wt cells, SGI-1776 had no effect on cell cycle at concentrations up to 5 μM, and caused apoptosis at 10 μM, while in FLT3-ITD cells, G1 arrest and apoptosis occurred at 100 nM. HL60 colony formation was completely inhibited by 5 μM SGI-1776, while MOLM-14 colony formation inhibition occurred at 500 nM. Finally, SGI-1776 sensitized multidrug resistant, but not parental, cells to multidrug resistance protein substrate, but not non-substrate, drugs. SGI-1776 at 1 μM decreased the IC₅₀ of the Pgp substrate chemotherapy drug daunorubicin in Pgp+ HL60/VCR cells 7-fold, but had no effect on daunorubicin IC₅₀ in HL60 cells, nor on IC₅₀ of the non-Pgp substrate cytarabine in either cell line. SGI-1776 at 1 μM also decreased the IC₅₀ of the BCRP substrate chemotherapy drug mitoxantrone in BCRP+ 8226/MR20 cells 7-fold. SGI-1776 at 1 μM doubled the percentage of apoptotic cells among HL60/VCR, but not HL60, cells exposed to daunorubicin and 8226/MR20 cells exposed to mitoxantrone. Finally, SGI-1776 at 1 μM decreased HL60/VCR colony formation in the presence of daunorubicin, but not cytarabine, but had no effect in HL60 cells, and also decreased 8226/MR20 colony formation in the presence of mitoxantrone, but not cytarabine. Thus the Pim-1 inhibitor SGI-1776 has anti-proliferative effects in AML cells with wt FLT3 as well FLT3-ITD, and sensitizes Pgp+ and BCRP+ multidrug resistant cells to chemotherapy. These data support clinical trials of SGI-1776 in AML with wt FLT3 as well FLT3-ITD, as a single agent and in combination with chemotherapy in multidrug resistant AML.