Simultaneous Sessions I: Mechanisms and strategies to address TKI resistance and stem cell persistence
In chronic myeloid leukaemia (CML), BCR-ABL tyrosine kinase inhibitors (TKI) fail to eliminate leukaemia stem cells (LSC) which can serve as a reservoir to drive relapse, TKI resistance and promote disease progression. Therefore, identification of pathways/targets that promote LSC survival is essential for the development of curative therapies. Our previous transcriptomics analysis pointed to neurotransmitter pathways as being aberrantly expressed in LSC compared to normal haemopoietic stem cells.
This led us to hypothesise that LSCs require neurotransmitter pathways to maintain their stem cell potential. The aim of our study was therefore to understand whether modulating these pathways would affect LSC survival.
To identify drug-able targets within these pathways, we performed compound screens in BCR-ABL-/+ cell lines and primary CML CD34+ cells using 658 compounds known to modulate neurotransmitter signalling, many of which are used clinically to treat neuro-psychiatric disorders. Hits selective for BCR-ABL-expressing cells were further evaluated in phenotypic assays using primary CML CD34+ versus non-CML CD34+ cells. Western blotting and FACs analysis was used to determine the effects that these compounds had on cellular pathways known to affect LSC survival.
3-CPMT, a selective dopamine reuptake inhibitor (SDRI), and paroxetine, a clinical grade selective serotonin reuptake inhibitor (SSRI), that target SLC6A3 and SLC6A4 respectively, emerged as compounds that selectively reduced CML CD34+ cell counts (P = 0.0007 and 0.04 respectively) and inhibited CFC outputs (P = 0.001 and 0.04, respectively). FACs analysis has shown that LSC and CML cell lines express 3-4-fold higher levels of SLC6A3 and SLC6A4 than their non-CML counterparts, with expression differences for SLC6A4 reaching significance (CML CD34+CD38- versus non-CML CD34+CD38- cells, P = 0.04; BaF3 BCR-ABL+ versus wild type BaF3, P = 0.04). Targeting of SLC6A3 or SLC6A4 with 3-CPMT or paroxetine +/- nilotinib inhibits a number of known LSC survival factors including PRKCH, FOXO3a, p38 MAPK and c-Myc. Furthermore, we have demonstrated that paroxetine in combination with nilotinib is highly effective at eradicating CD34+CD38-CD90+ LSC compared to nilotinib alone in patient-derived xenograft mouse models (P = 0.01).
In conclusion, our pre-clinical data suggest that repurposing drugs that target neurotransmitter transporters may represent a novel way to target LSC in CML patients.