The pan-Bcl2 Inhibitor AT101 Activates the Intrinsic Apoptotic Pathway and Causes DNA Damage in Acute Myeloid Leukemia Stem-Like Cells

Abstract

Background

Leukemia stem cells (LSCs) are considered to be the cause of treatment failure and relapse in acute myeloid leukemia (AML). Overexpression of the Bcl-2 family of anti-apoptotic proteins such as Bcl-2, Bcl-xl, and Mcl-1 accounts for survival and self-renewal of LSCs. AT101 binds to the BH3 motif of all Bcl-2 family anti-apoptotic proteins and demonstrates anti-tumor activity in multiple types of tumor. Thus, we hypothesized that this agent might have the potential to deplete LSCs.

Objective

The present study aims to investigate if and by what mechanism AT101 is able to target AML stem-like cells.

Methods

As LSCs and hematopoietic stem cells (HSCs) are enriched in CD34⁺CD38⁻ populations, CD34⁺CD38⁻ cells from KG1α and Kasumi-1 cell lines as well as CD34⁺ blasts from AML patients were used as LSC models, while CD34⁺ cells from healthy donors were used as normal hematopoietic cells. Cell proliferation and apoptosis were assessed by a cell counting kit-8 (CCK8) assay and an Annexin V/PI assay using flow cytometry, respectively. Colony-forming units experiments were performed to monitor the stemness features of AML cells. Western blot and quantitative real-time polymerase chain reaction (qPCR) analysis were performed to examine the levels of proteins and mRNAs related to either the intrinsic apoptotic pathway or DNA damage response.

Results

AT101 inhibited proliferation and induced apoptosis in CD34⁺CD38⁻ KG1α and Kasumi-1 cells in a dose- and time-dependent manner. Exposure to AT101 for 24 h resulted in apoptosis in primary CD34⁺ AML blasts (EC₅₀ [concentration needed for a 50% maximal effect] = 2.45–76.00 μmol/L), while it only had a modest effect on normal CD34⁺ hematopoietic cells. Mechanistically, AT101 activated the intrinsic apoptotic pathway by inhibition of Bcl-2 anti-apoptotic proteins, reflected by a decrease in mitochondrial membrane potential. Moreover, AT101 caused DNA damage (e.g., increased γH2AX phosphorylation), which might also contribute to its anti-leukemic effects. Interestingly, the ex vivo efficacy of AT101 in primary AML samples significantly correlated to hyperleukocytosis and FLT3-ITD mutations. AT101 was also effective against CD34⁺ blasts isolated from elderly patients and patients who did not achieve complete remission after induction therapy.

Conclusions

AT101 effectively eliminates LSCs in vitro through the induction of DNA damage and activation of the intrinsic apoptotic pathway. AT101 is effective towards leukemic cells from patients with adverse prognostic factors, suggesting that AT101 could have the potential as an alternative salvage therapy for the treatment of relapsed and refractory AML.

http://ift.tt/2ugLVFJ