Abstract
Abstract 2163
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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
Disclosures:
Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.
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