Abstract
Acute myeloid leukemia (AML) is a serious disease and despite an improved understanding of the biology of AML, little has changed in the treatment of the disease over the last four decades. The 5 year survival rates of AML remain frustratingly low. Resistance to frontline chemotherapy remains the main cause of treatment failure; therefore a new treatment modality is urgently needed.
Bcl-2 is a promising therapeutic target; it is overexpressed in both bulk AML cells and leukemia-initiating cells. The Bcl-2-selective inhibitor ABT-199 (Venetoclax) shows promising anti-leukemic activity in patients with high-risk relapsed/refractory AML or unfit for intensive chemotherapy. However, resistance through another Bcl-2 family protein, Mcl-1, was shown by our lab and others to limit the anti-leukemic activity of ABT-199. Previous studies from our lab have shown that ABT-199 monotherapy decreases the association of Bim with Bcl-2 but is compensated for by its increased association with Mcl-1. We also found that ABT-199 increases cellular Mcl-1 levels by extending the protein's half-life. Based on these previous studies we tested the efficacy of direct inhibition of Bcl-2 by ABT-199 and Mcl-1 by A-1210477. ABT-199 and A-1210477 synergistically induced apoptosis in both ABT-199-resistant and sensitive AML cells. This combination treatment disrupted binding of Bim with Bcl-2 and Mcl-1 concurrently, with no observed binding of Bim to Bcl-xL. This work has demonstrated that targeting both Bcl-2 and Mcl-1 is necessary for AML treatment. However, our work also indicated that direct targeting of Mcl-1 with A-1210477 to enhance the anti-leukemic activity of ABT-199 might cause increased toxicity. Thus, indirect targeting of Mcl-1 may represent a better approach to enhance the anti-leukemic activity of ABT-199 in AML.
It was recently reported that targeting exportin 1 (XPO1), a nuclear exporter overexpressed in AML cells, selectively targets leukemia-initiating cells and can decrease Mcl-1 levels in cancer cells, though the underlying mechanism is unknown. KPT-330 (Selinexor) is a XPO1-selective inhibitor which is currently being tested in clinical trials for AML. Preliminary results have shown that KPT-330 induces a response at well-tolerated doses in AML and other hematological malignancies. Thus, XPO1 inhibition may represent a viable strategy for downregulating Mcl-1 to synergize with ABT-199 treatment in AML.
To test our hypothesis we tested various concentrations of ABT-199 and KPT-330 alone and in combination in AML cell lines and primary patient samples. At 24 h, synergistic induction of apoptosis was observed between the two drugs in THP-1, CTS, Molm-13, and OCI-AML3 AML cell lines. Synergy was also found in 4 relapsed and 6 newly diagnosed primary AML patient samples. To test the effect of drug treatment on leukemia-initiating cells, we performed colony formation assays on primary AML patient samples post drug treatment and found that combination treatment significantly enhanced inhibition of colony formation when compared to single drug treatment, demonstrating that KPT-330 combined with ABT-199 is effective in targeting the leukemic bulk and the leukemia-initiating cells.
To determine the mechanism by which the combination works, we measured the protein levels of various Bcl-2 family members. Decreased protein levels for Mcl-1 and surprisingly Bim were observed post KPT-330 or KPT-330 plus ABT-199 treatment, while protein levels for Bcl-2 and Bcl-xL remained unchanged. A time course experiment revealed that Mcl-1 levels decreased at apoptosis initiation while Bim levels decreased after apoptosis initiation, suggesting that Mcl-1 plays a critical role in apoptosis induced by the drug combination, while Bim may not. Mcl-1 knockdown enhanced combination treatment of ABT-199 and KPT-330. Reciprocal Mcl-1 overexpression partially rescued the cells from drug treatment but was incomplete, likely due to Mcl-1 overexpression not being high enough and/or non-Mcl-1 pathways through which KPT-330 acts. In summary, Mcl-1 is modulated by KPT-330 treatment alone and in combination with ABT-199 and this modulation of Mcl-1 plays an important role in initiation of apoptosis in response to treatment. Additional studies are underway to further elucidate the molecular mechanisms of combination ABT-199 and KPT-330 treatment and to determine the in vivo anti-leukemic efficacy in NSG mouse AML models.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
