Asciminib (previously ABL001), which binds the myristate-binding pocket of the 
Bcr-Abl kinase domain, is in phase I clinical trials as monotherapy and in 
combination with imatinib, nilotinib and dasatinib for the treatment of patients 
with refractory CML or Ph+ ALL. Asciminib sensitivity was evaluated in asciminib 
naïve BCR-ABL1+ cell lines K562 (negligible ABCB1/ABCG2 expression), K562-Dox 
(ABCB1-overexpressing through doxorubicin exposure) and K562-ABCG2 (ABCG2 
overexpression via transduction) with results demonstrating asciminib efflux by 
both ABCB1 and ABCG2 transporters. K562-Dox and K562-ABCG2 cells demonstrated 
increased LD50asciminib vs K562 control cells: 256 and 299 nM respectively vs 24 
nM, p < 0.001. Sensitivity was completely restored with specific inhibitors 
cyclosporine (ABCB1) and Ko143 (ABCG2): K562-Dox LD50asciminib+cyclosporine = 13 
nM, K562-ABCG2 LD50asciminib+Ko143 = 15 nM (p < 0.001). When asciminib 
resistance was modelled in vitro, ABCB1 and ABCG2 overexpression was integral in 
the development of asciminib resistance. In K562 asciminib-resistant cells, 
ABCG2 expression increased prior to BCR-ABL1 overexpression and remained high 
(up to 7.6-fold greater levels in resistant vs control cells, p < 0.001). 
K562-Dox asciminib-resistant cells had increased ABCB1 expression (2.1-fold vs 
control cells p = 0.0033). KU812 asciminib-resistant cells overexpressed ABCB1 
(5.4-fold increase, p < 0.001) and ABCG2 (6-fold increase, p < 0.001) before 
emergence of a novel myristate-binding pocket mutation (F497L). In all three 
cell lines, asciminib resistance was reversible upon chemical inhibition of 
ABCB1, ABCG2 or both (p < 0.001). When K562 asciminib-resistant cells were 
treated with asciminib in combination with clinically achievable doses of either 
imatinib or nilotinib, reversal of the resistance phenotype was also observed (p 
< 0.01). Overexpression of efflux transporters will likely be an important 
pathway for asciminib resistance in the clinical setting. Given the lack of 
evidence for ABCG2-mediated transport of nilotinib or imatinib at clinically 
relevant concentrations, our data provide an additional rationale for using 
asciminib in combination with either TKI.