The FLT3 Inhibitor PKC412 Interacts Synergistically with Both Daunorubicin and Cytarabine in Acute Myeloid Leukemia (AML) Cells by Heterogeneous Mechanisms.

Both FLT3 activating mutations and overexpression of ATP-binding cassette (ABC) transport proteins that confer multidrug resistance (MDR) are associated with inferior treatment outcomes in AML. The protein kinase C inhibitor PKC412 inhibits both FLT3 signaling and drug transport by the ABC protein P-glycoprotein (Pgp; ABCB1). The effects of PKC412 on other MDR-associated ABC proteins expressed in AML cells, including multidrug resistance protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; ABCG2), are not known. We studied the effects of PKC412 on transport of MDR protein substrate drugs and its interactions with the MDR protein substrate daunorubicin (DNR) and the non-MDR protein substrate cytarabine (AraC) in cell lines overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20), in wild-type HL60 cells, which express no MDR proteins, and in 8 AML patient bone marrow samples characterized for FLT3 mutations by a DNA-based assay and for expression and function of Pgp, MRP-1 and BCRP by flow cytometry using specific antibodies (MRK16, MRPm6 and BXP21) and modulators (PSC-833, probenecid and fumitemorgin C). in vitro drug sensitivity was assessed by cell survival measured by the wst-1 assay following 48- (AML samples) or 96- (cell lines) hour continuous drug exposure in 96-well microculture assays, and drug interactions were evaluated by the combination index approach of Chou and Talalay. PKC412 increased uptake of MDR protein substrate drugs in cell lines overexpressing MRP-1 and BCRP, as well as Pgp, in a concentration-dependent manner, with maximum effects at ≥5 μM. PKC412 was cytotoxic to all cell lines studied, and cell lines overexpressing Pgp, MRP-1 and BCRP were 20- to 40-fold resistant to PKC412 in relation to parental drug-sensitive cells, suggesting that PKC412 may be a substrate for Pgp, MRP-1 and BCRP and may thus modulate drug transport by competitive inhibition. The combination of PKC412 with DNR synergistically decreased cell survival (CI₅₀ values <0.9) in cell lines overexpressing Pgp, MRP-1 and BCRP, but not in wild-type HL60 cells, whereas the combination of PKC412 and AraC synergistically decreased cell survival in wild-type HL60 cells as well as in the cell lines expressing Pgp, MRP-1 and BCRP. These data suggest that PKC412 interacts synergistically with the MDR protein substrate drug DNR at least in part by virtue of modulation of its transport, but interacts with AraC through alternative mechanisms in both wild-type and multidrug resistant cells. The eight AML patient samples studied included 4 each with FLT3 internal tandem duplication (ITD) and wild-type FLT3, and in vitro sensitivity to PKC412 did not differ between these two groups. All eight samples exhibited expression or function of at least one ABC transport protein. Synergistic cytotoxicity was observed for the combination of PKC412 with DNR in 7 of 8 AML patient samples, whereas the combination of PKC412 with AraC produced synergistic cytotoxicity in 4, including 3 with wild-type FLT3 and one with FLT3 ITD. Thus combinations of PKC412 with DNR and with AraC have the potential for synergistic activity in the treatment of AML, and synergy occurs both through modulation of Pgp, MRP-1 and BCRP and through other, likely heterogeneous, mechanisms. Interactions of PKC412 with both MDR protein substrate and non-substrate chemotherapy drugs have the potential to be exploited in novel combination regimens.