Erlotinib Inhibits ABC Transporters of AML Progenitors with Stem Cell Features and Increases Chemosensitivity to Current AML Drugs

Treatment failure in AML is attributed to the persistence of AML progenitors able, among others, to efflux chemotherapeutic drugs via ABC-transporters. Increased efflux capacity is considered a stem cell feature, and therapeutic inhibition may increase chemosensitivity and help eradicate this progenitor population. Nevertheless, clinical studies assessing a potential benefit of ABC-inhibitors in AML treatment showed no significant survival advantage, possibly because AML cells express different ABC-transporters and classical inhibitors target only a restricted type of efflux channels. We assessed the efficacy of the TKI erlotinib (Erlo) to antagonize drug efflux via most important AML-associated efflux channels, ie P-gp, MRP and BCRP.

Overall drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ), and specific efflux via P-gp (substrates: DioC₂³ and rhodamine-123), MRP (substrates: calcein and CDCFDA) and BCRP (substrate: Hoechst 33342) were quantified by FACS at 1h and 6h following incubation with 10mM Erlo. Biochemical inhibitors of the respective ABC-transporters (CSA, verapamil, MK-571, KO143) served as controls. Surface expression of P-gp, MRP and BCRP was quantified by FACS. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), doxorubicine (Dox, 100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC₃(6)/PI staining. Assays were carried out in myeloid cell lines (KG-1, MOLM-13, HL-60) and ex vivo AML cells (n=3). Immaturity of AML cells was determined in 2 samples by comparing CD34+ versus CD34- cells, and in one pt by co-staining for CD34, CD38, CD123 and CD133.

We found that I) Erlo inhibited efflux via P-gp and MRP as demonstrated by increased intracellular retention of DioC₂³/Rho-123, and calcein/CDCFDA, respectively; II) this degree of inhibition was higher in KG-1 cells than in MOLM-13 or HL-60 cells; III) inhibition of drug efflux was observed already at 1h of incubation, increased over time (6h); IV) Erlo increased intracellular retention of MTZ faster (at 1h with a further increase at 6h) and at least to the same extent than a combination of all three biochemical efflux inhibitors, showing that Erlo's capacity to hinder drug efflux is not restricted to a single ABC-transporter: V) surface expression of P-gp, MRP and BCRP was strongest on KG-1 cells and not altered upon 1h and 6h of Erlo incubation VI) Erlo increased Dox- and VP16-induced apoptosis (48h KG-1: Erlo alone 20%, Dox alone 10%, VP-16 alone 20%, Erlo+Dox: 40%, VP-16+Erlo: 70%), while having no impact on AraC-induced apoptosis; VI) this pattern of chemosensitization was observed in all myeloid cell lines, but once more most pronounced in KG-1 cells. To test the hypothesis that Erlo has comparable effects in pt-derived AML cells ex vivo, we showed by concomitant cell surface staining that I) immature AML subpopulations had a higher efflux capacity (notably via P-gp) than their more mature counterparts (i.e. in one pt with chemoresistant AML: DioC₂³/Rho-123 fluorescence twice as high in the CD34-/CD38+, CD123+, CD133- than in the CD34+/CD38dim, CD123-, CD133+ subpopulation); II) cell surface expression of P-gp is twice as high in this more immature population (CD34+/CD38dim, CD123-, CD133+) than in CD34-/CD38+, CD123+, CD133+ cells; III) Erlo antagonizes drug efflux via P-gp and MRP at 1h (increasing further at 6h) of incubation; IV) this effect is most pronounced in the immature progenitor cells (1h: decrease of DioC₂³/Rho-123 efflux in CD34-/CD38+, CD123+, CD133- cells by about 50% and in the more immature CD34-/CD38+, CD123-, CD133+ cells by about 70%); V) Erlo diminishes cell surface expression of P-gp (48h), most effectively in the progenitor populations (by 30% in the CD34-/CD38+, CD123+, CD133- cells versus 50% in CD34-/CD38+, CD123+, CD133- cells); VI) Erlo is able to retain MTZ in both CD34- and CD34+ AML-subpopulations; VII) these effects are accompanied by an increased sensitivity towards Dox and VP-16; VIII) Erlo-induced chemosensitization is higher in the CD34+ than in CD34- AML cells.

We here provide novel evidence that erlotinib is able to overcome the stem cell features of increased expression and functionality of ABC-transporters thereby antagonizing the intrinsic chemoresistance of (immature) AML cells. Those results suggest a potential clinical interest of combining erlotinib to chemotherapy in AML

Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.