Amonafide L-Malate Bypasses Multidrug Resistance Proteins in Secondary Acute Myeloid Leukemia.
Authors: O’Loughlin Kieran L. , Minderman Hans , Ford Laurie A. , Baer Maria R. 
Published: April 2020

Abstract
Amonafide L-malate, a DNA intercalating agent and ATP-independent topoisomerase 2 inhibitor showed promising clinical activity in combination with cytarabine in a Phase 2 clinical trial in secondary acute myeloid leukemia (S-AML), with a complete remission (CR) rate of 45% (Erba, et al., JCO25:373s, 2007), and is currently in a Phase 3 clinical trial. We hypothesized that the activity of amonafide L-malate in S-AML may be attributable in part to its being a poor substrate for the multidrug resistance (MDR)-associated drug efflux proteins expressed in AML cells, including P-glycoprotein (Pgp; MDR1; ABCB1), multidrug resistance protein-1 (MRP-1;ABCC1) and breast cancer resistance protein (BCRP;ABCG2). Amonafide has been previously shown not to be a substrate for Pgp in cell lines overexpressing this protein (Chau, et al., Leuk Res, in press). Here we studied transport and cytotoxicity of amonafide L-malate and its active metabolite N-acetyl amonafide in cell lines overexpressing Pgp, MRP-1 and BCRP and in pretreatment blasts from patients with S-AML. Cellular drug transport was studied by flow cytometry and cytotoxicity was studied in 96-well microcultures in the absence and presence of the MDR modulators PSC-833 (Pgp), probenecid (MRP-1) and fumitremorgin C (FTC), and were compared with those of classical topoisomerase 2 inhibitors including doxorubicin, daunorubicin, idarubicin, mitoxantrone and etoposide in leukemia and myeloma cell lines overexpressing Pgp, MRP-1 and BCRP and in pretreatment marrow samples from 22 S-AML patients characterized with regard to expression and function of these proteins. S-AML patients included 9 with therapy-related AML and 13 with antecedent myelodysplastic syndromes. Pgp, MRP-1 and BCRP expression in pretreatment blasts was measured by flow cytometry with the MRK16, MRPm6 and BXP21 antibodies, and function by uptake of the fluorescent substrates DiOC2(3), rhodamine-123 and pheophorbide A with modulation by PSC-833, probenecid and FTC, respectively. Uptake, efflux and cytotoxicity of amonafide L-malate did not differ in resistant cell lines overexpressing Pgp, MRP-1 and BCRP, compared to parental cells, and did not differ in the presence and absence of MDR modulators, in contrast to all of the other topoisomerase 2 inhibitors studied. Cell lines overexpressing Pgp and MRP-1 did not display transport of, or resistance to, the metabolite, acetyl amonafide, but acetyl amonafide was a substrate for BCRP, with 8-fold resistance in cells with BCRP expression, and 8-fold sensitization by FTC. Pgp, MRP-1 and BCRP expression and/or function was observed in 18, 7 and 17 of 22 secondary AML samples, respectively. Cyclosporin A, which inhibits substrate drug efflux by Pgp, MRP-1 and BCRP, increased uptake of daunorubicin, idarubicin and amonafide L-malate by mean values of 19.7%, 7% and −2.5%, respectively, and increased uptake by ≥ 10% in 16, 12 and 5 patient samples. In conclusion, in relation to other topoisomerase 2 inhibitors used to treat AML, including daunorubicin, idarubicin, mitoxantrone, and etoposide, amonafide L-malate is a poor substrate for the MDR proteins expressed in AML cells in general, and S-AML cells in particular. The encouraging complete remission rates seen with amonafide L-malate treatment in clinical trials in S-AML may thus be attributed at least in part to its potential to overcome MDR in S-AML cells.