MTH1 Inhibitor-Induced Cytotoxicity in Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%.  The major reason for treatment failure in AML is resistance to chemotherapy.  Thus, there is an urgent need for identification of novel therapeutic agents for AML.

Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS).  Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death.  MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA.  MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death.  Whether MTH1 inhibitors have any activity against AML is not known.

METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours.  In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588.  Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry.  To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml).  Percentage apoptosis was calculated by normalizing to vehicle only control.

With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls.  Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control.

RESULTS:  TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL.  In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC₅₀ 3.1-21.3µM, Figure 1).  Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death.  In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTracker^Low cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC₅₀ 6µM, Figure 3). Analysis of additional primary AML samples is ongoing.

DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells.  MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells.  In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine.  Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted.

Figure 1: Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. 

Figure 2: TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine.

Figure 3: TH588 significantly diminished colony formation in primary AML ex vivo in dose-dependent manner.