The Allosteric Inhibitor ABL001 Is Susceptible to Resistance in Vitro Mediated By Overexpression of the Drug Efflux Transporters ABCB1 and ABCG2
Authors: Eadie Laura N , Saunders Verity A , Leclercq Tamara M , Branford Susan , White Deborah L , Hughes Timothy P. 
Published: October 2019

Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2.

ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once >80% survival in culture for >10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed.

K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p<0.001 suggesting both ABCB1 and ABCG2 are important in ABL001 transport. Furthermore, resistance was reversible through use of specific inhibitors cyclosporin (ABCB1) and Ko143 (ABCG2). IC50ABL001 +inhibitors was comparable to that of control K562 cells: K562-Dox +cyclosporin=11 nM; K562-ABCG2 +Ko143=15 nM.

A prior study identified ABCB1 overexpression as an initiator of resistance to IM and NIL (expression increased up to 7- and 5-fold in IM and NIL resistant cells compared with respective control cells p<0.002). In this study, expression levels of ABCB1 and ABCG2 were interrogated in 3 ABL001 resistant cell lines; results indicated overexpression of both transporters was integral in development of resistance. Up to 17- and 60-fold greater levels of ABCB1 and ABCG2 respectively was observed in resistant vs control cells (Table 1). No KD mutations were detected in early resistance intermediates; however, a myristate pocket mutation was detected in later stage KU812 ABL001 resistant cells (percentage mutation correlated with IC50ABL001).

Table 1. ABL001 resistance characteristics in selected resistant intermediates IC50ABL001 (nM) Fold change (MFI) Bcr-Abl over expression KD mutation ABCB1 ABCG2 K562 Control 23 N N K562 500 nM ABL001 >2500 p <0.001 -1.4 p =0.003 48.3 p =0.007 Y N K562 10 μM ABL001 27, 800p <0.001 -2.3 p =0.535 60.3 p <0.001 Y N K562 10 μM ABL001 +Ko143 89 p <0.001 K562-Dox Control 256 N N K562-Dox 500 nM ABL001 2393 p <0.001 2.3 p <0.001 -2.9 p <0.001 N N K562-Dox 500 nM ABL001 +cyclosporin 17 p <0.001 K562-Dox 10 μM ABL001 >50, 000 p <0.001 -1.6 p <0.001 -0.1 p <0.001 N N KU812 Control 2.7 N N KU812 5 nM ABL001 6.4 p <0.001 2.2 p =0.008 4.0 p =0.002 Y N KU812 10 μM ABL001 33, 300 p <0.001 8.1 p =0.003 11.0 p =0.010 Y Y (90%) p-value: resistant vs respective control; n>3; N=No; Y=Yes

While further in vitro and in vivo studies will determine the clinical relevance of efflux-mediated resistance to ABL001 mono- and combination therapy, our preclinical data provide evidence that kinase domain mutations may not be the predominant cause of ABL001 resistance; drug transporters likely play an important role as well. Susceptibility to ABCB1 overexpression is well recognised for IM and NIL resistance and we now show it is also relevant for ABL001. ABCG2-mediated resistance has not been observed with NIL or IM but is clearly important for ABL001. ABL001 treatment induced ABCG2 overexpression in two cell lines; both have negligible basal expression. ABCG2 overexpression preceded Bcr-Abl overexpression and mutation emergence. Importantly, ABL001 resistance was completely reversible in the presence of the ABCG2 inhibitor Ko143 (K562 10 μM ABL001 cells). Given the lack of strong evidence for ABCG2-mediated transport of NIL or IM at clinically relevant concentrations, our data provide a strong rationale for the use of ABL001 in combination with either of these TKIs.

Disclosures

Branford: Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.