Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec 27;7(52):86186-86197.
doi: 10.18632/oncotarget.13364.

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia

Affiliations

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia

Claudia M Kowolik et al. Oncotarget. .

Abstract

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by heterogeneous genetic and epigenetic changes in hematopoietic progenitors that lead to abnormal self-renewal and proliferation. Despite high initial remission rates, prognosis remains poor for most AML patients, especially for those harboring internal tandem duplication (ITD) mutations in the fms-related tyrosine kinase-3 (FLT3). Here, we report that a novel epidithiodiketopiperazine, NT1721, potently decreased the cell viability of FLT3-ITD+ AML cell lines, displaying IC50 values in the low nanomolar range, while leaving normal CD34+ bone marrow cells largely unaffected. The IC50 values for NT1721 were significantly lower than those for clinically used AML drugs (i.e. cytarabine, sorafenib) in all tested AML cell lines regardless of their FLT3 mutation status. Moreover, combinations of NT1721 with sorafenib or cytarabine showed better antileukemic effects than the single agents in vitro. Combining cytarabine with NT1721 also attenuated the cytarabine-induced FLT3 ligand surge that has been linked to resistance to tyrosine kinase inhibitors. Mechanistically, NT1721 depleted DNA methyltransferase 1 (DNMT1) protein levels, leading to the re-expression of silenced tumor suppressor genes and apoptosis induction. NT1721 concomitantly decreased the expression of EZH2 and BMI1, two genes that are associated with the maintenance of leukemic stem/progenitor cells. In a systemic FLT3-ITD+ AML mouse model, treatment with NT1721 reduced tumor burdens by > 95% compared to the control and significantly increased survival times. Taken together, our results suggest that NT1721 may represent a promising novel agent for the treatment of AML.

Keywords: BMI1; DNMT1; ETP; FLT3 ligand; FLT3-ITD.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

Disclosure of Potential Conflicts of Interest: DAH and LEO are cofounders of Novonco Therapeutics Inc.

Figures

Figure 1
Figure 1. Effect of NT1721 on the viability of primary AML samples and normal CD34+ bone marrow cells
(A) Chemical structure of NT1721. (B) The cell viability of normal CD34+ bone marrow cells and primary AML samples from peripheral blood (PB) or bone marrow (BM) of two newly diagnosed AML patients was determined after 48 h of treatment with NT1721. The graphs represent the mean ± SD from three experiments. The asterisks indicate statistical significant differences compared to the untreated control (P values < 0.05). (C) Apoptosis induction. Primary AML cells from bone marrow were treated with NT1721 for 48 h and stained with annexin V.
Figure 2
Figure 2. Drug combinations of NT1721 with cytarabine or sorafenib display better antileukemic properties than the single agents
(A) Apoptosis induction. Molm14 cells were treated for 48 h with the single agents or the drug combinations as indicated, stained with annexin V and analyzed by flow cytometry. The graphs represent the mean ± SD from three independent experiments. The asterisks indicate statistical significant differences compared to the respective single agents (P values < 0.01). (B and C) Cell cycle analysis. Molm14 cells were treated for 48 h, fixed, stained with PI and subjected to flow cytometry; the data were analyzed with FlowJo software using the Watson model.
Figure 3
Figure 3. Treatment with NT1721 led to the depletion of DNMT1, EZH2 and BMI1 and induced the expression of tumor suppressor genes
Molm14 were treated with NT1721 as indicated for 24 h. (A) Western blot analysis of EZH2, DNMT1, and BMI1 expression. (B) QPCR analysis of DNMT1, DNMT3B and BMI1 expression. The data were analyzed using GAPDH as reference gene. The graphs represent the mean ± SD from at least three independent experiments. The asterisks indicate statistical significant differences compared to the untreated controls (P values < 0.05). (C) QPCR analysis of the expression of tumor suppressor genes, CDKN2B (p16) and BIM. The graphs represent the mean ± SD from three independent experiments. The asterisks indicate statistical significant differences compared to the untreated control (P values < 0.05). (D) Western blot analysis of CDKN2A (p16) and BIM expression.
Figure 4
Figure 4. NT1721 attenuated the cytarabine-induced FLT3LG upregulation
Molm14 were treated with NT1721 and cytarabine as indicated. (A and B) qPCR analysis of FLT3LG expression.The data were analyzed using GAPDH as reference gene. The graphs represent the mean ± SD from three independent experiments. The asterisks indicate statistical significant differences compared to the untreated controls (P values < 0.01). (C) FACS analysis of FLT3LG expression in Molm14 cells.
Figure 5
Figure 5. Efficacy of NT1721 in a systemic AML mouse model
NSG mice were injected (I.V.) with 106 luc+ Molm14 cells. Four days later mice were divided into groups bearing equal tumor burdens and treated by gavage with the indicated doses of NT1721 or the vehicle control. (A) In vivo monitoring of bioluminescent signals. Female (F) and male (M) mice were treated as indicated. Bioluminescent signals were monitored on the indicated days and expressed as photons/s/mm2 ± SD. (B) Bioluminescent signals in male NSG mice. Two representative mice from each treatment group are shown. The color bars show the relative luciferase activity in 106 photons/s/mm2. (C) Survival curves. Groups of female and male NSG mice (at least 3 per group) were treated with the indicated doses of NT1721 or the vehicle control until they died or met the criteria for euthanasia. (D) Changes in gene expression in vivo. Human cells were isolated from the bone marrow of individual mice after 14 days of treatment (daily dose of 15 mg/kg of NT1721 or vehicle, n = 3/group). Purified total RNA from individual mice was used for qPCR assays. The graphs represent the mean ± SD. The asterisks indicate statistical significant differences compared to the control group (P values < 0.01).

References

    1. Kayser S, Levis MJ. FLT3 tyrosine kinase inhibitors in acute myeloid leukemia: clinical implications and limitations. Leuk lymphoma. 2014;55:243–255. - PMC - PubMed
    1. Swords R, Freeman C, Giles F. Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia. Leukemia. 2012;26:2176–2185. - PubMed
    1. Ramos NR, Mo CC, Karp JE, Hourigan CS. Current Approaches in the Treatment of Relapsed and Refractory Acute Myeloid Leukemia. J Clin Med. 2015;4:665–695. - PMC - PubMed
    1. Robak P, Robak T. Older and new purine nucleoside analogs for patients with acute leukemias. Cancer Treat Rev. 2013;39:851–861. - PubMed
    1. Ravandi F, Kantarjian H, Faderl S, Garcia-Manero G, O'Brien S, Koller C, Pierce S, Brandt M, Kennedy D, Cortes J, Beran M. Outcome of patients with FLT3-mutated acute myeloid leukemia in first relapse. Leuk Res. 2010;34:752–756. - PMC - PubMed