Molecular and Pharmacological Inhibition of ATP Binding Cassette Transporter ABCB6 Decreases Chemoresistance in Acute Myeloid Leukemia

Cytarabine (Ara-C) and Daunorubicin (Dnr) remain the main stay treatment for patients with acute myeloid leukemia (AML). Drug resistance and relapse are the major hurdles in achieving long term event-free survival. ATP Binding Cassette transporter proteins (ABC) plays a prominent role in drug resistance by extruding the anticancer drugs from the cells. Among the mitochondrial ABC transporters, the heme protoporphyrin transporter ABCB6 was found to be highly expressed in cell lines- HT-29, A549, SK3, St-4, A2780, SKOV3, KK47, T24, K562, and U937 made resistant to camptothecin, cisplatin, etoposide (VP-16), adriamycin and also in HepG2 and Hep3B cell lines resistant to arsenite. We have previously reported that ABCB6 expression was significantly higher in primary AML samples resistant to Dnr (Varatharajan et al., 2017). AML patients with FLT3 -ITD showed low ABCB6 expression levels and were more sensitive to arsenic trioxide (ATO) compared to FLT3 wild type (Abraham et al., 2014). In the present study, using a molecular and inhibitor based approach we explored the functional role of ABCB6 in mediating resistance to Dnr and ATO in AML.

Cell lines K562 and THP1 with high ABCB6 expression and high IC50 to both Dnr and ATO were transduced with an inducible lentiviral shRNA targeting ABCB6 . The efficiency of knock down was confirmed by real time PCR, immunoblot and immunofluorescence. As ABCB6 is known to localize in the mitochondrial membrane, its role in mediating reactive oxygen species (ROS) levels were assessed by flowcytometry using DCFDA. The effect of ABCB6 knock down in causing apoptosis to Dnr and ATO mediated apoptosis was evaluated using 7AAD and Annexin V. The effect of ABCB6 pharmacological inhibitor, tomatidine in improving chemosensitivity was also evaluated in combination with Dnr and ATO by MTT assay. The efficiency of knock down was >80% in K562 (p<0.001) and >60% in THP1 (p<0.001) cell lines. Concordant to the mRNA levels, there was a significant decrease in ABCB6 protein levels by western blot and immunofluorescence. Interestingly, ABCB6 knock down increased basal ROS levels in both K562 (18 fold) and THP1 (3.1 fold) compared to their respective doxycycline negative (Dox -ve) control. The combination of ABCB6 knockdown and Dnr treatment increased ROS by 2.1-fold in THP1 and by 21-fold in K562 cells and along with ATO by 1.7-fold in THP1 and 5.7-fold in K562 compared to the Dox -ve cells. Knockdown of ABCB6 also improved sensitivity to 0.125µM of Dnr (17.45% to 36.94%, p= ns) and 6µM ATO (10% to 39%, p=0.007). ABCB6 inhibitor tomatidine reduced the IC50 values of THP1 to Dnr (0.16µM to 0.05µM) and ATO (>6µM to 2.91µM) and K562 cells to ATO (12.08µM to 7.26µM). Our results suggest that targeting ABCB6 could sensitize different subgroups of AML to treatment with ROS inducing agents like Dnr and ATO. Further mechanistic studies are required to investigate if the effect of ABCB6 inhibition is selective to leukemic cells.