In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells

Justin J Montoya,Daniel H Wai,Vijay Gokhale,David W Lee,Megan A Turnidge,Apurvi R Patel,David O Azorsa,Laurence H Hurley,Robert J Arceci,Cynthia Wetmore

doi:10.1186/s12885-019-6464-9

Abstract

BackgroundAcute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be “undrugable” because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription.MethodsThis study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt’s lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents.ResultsGQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin.ConclusionThese results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

Highlights

Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate
GQC-05 induces apoptosis in AML cells AML cell lines KG-1a, CMK, and TF-1 were treated with GQC-05 for 24 h and analyzed for induction of caspase 3/7 as an indication of apoptosis
Increased annexin V staining in was induced by treatment with GQC-05 with the greatest increase in KG-1a cells (Fig. 2b), suggesting that KG-1a cells are more sensitive to the effects of GQC-05

Summary

Introduction

Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Genomic analysis has demonstrated that AML is composed of a heterogeneous population of malignant cells with numerous genetic complexities [2, 3] These genetic aberrations often lead to dysregulation of proto-oncogenes such as CREB, MYC, EVi-1, MN-1 [4, 5], resulting in difficult to treat tumor cells and poor clinical outcome [6]. G-quadruplexes (G4) are secondary four-stranded helical structures of DNA comprised of guanine-rich nucleic acids that are enriched in the promoter regions of many genes. One of the first reports that G4 s near promotor regions can effect gene expression arose from studies of the c-MYC oncogene, demonstrating that mutation of the endogenous G4 sequence or addition of a G4-stabilizing ligand altered transcription in vivo [10]

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