Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma

Abstract

Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of debilitating, incurable malignancies. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes, accounting for ~65% of CTCL cases. Patients with advanced disease have a poor prognosis and low median survival rates of four years. CTCLs develop from malignant skin-homing CD4⁺ T cells that spread to lymph nodes, blood, bone marrow and viscera in advanced stages. Current treatments options for refractory or advanced CTCL, including chemotherapeutic and biological approaches, rarely lead to durable responses. The exact molecular mechanisms of CTCL pathology remain unclear despite numerous genomic and gene expression profile studies. However, apoptosis resistance is thought to play a major role in the accumulation of malignant T cells. Here we show that NT1721, a synthetic epidithiodiketopiperazine based on a natural product, reduced cell viability at nanomolar concentrations in CTCL cell lines, while largely sparing normal CD4⁺ cells. Treatment of CTCL cells with NT1721 reduced proliferation and potently induced apoptosis. NT1721 mediated the downregulation of GLI1 transcription factor, which was associated with decreased STAT3 activation and the reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-xL). Importantly, NT1721, which is orally available, reduced tumor growth in two CTCL mouse models significantly better than two clinically used drugs (romidepsin, gemcitabine). Moreover, a combination of NT1721 with gemcitabine reduced the tumor growth significantly better than the single drugs. Taken together, these results suggest that NT1721 may be a promising new agent for the treatment of CTCLs.

Keywords: GLI1; NT1721; STAT3; cutaneous T cell lymphoma (CTCL); epidithiodiketopiperazine.

Figure 1

NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and HH cells were treated with increasing concentrations of NT1721. Cell viability and IC₅₀ values were determined after 48 and 72 h. (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4⁺ cells were treated with 300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D) Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The graphs represent the mean ± SD from triplicate values.

Figure 2

NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤ 0.05; ** indicates p ≤ 0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4⁺ cells, CTCL cells and NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4.

Figure 3

NT1721 induced apoptosis in CTCL cells in vitro and in vivo. (A) HuT78 cells were treated with NT1721 for 24 h or 48 h, then stained with annexin V and subjected to FACS analysis. (B) HuT78 and HH cells were treated with NT1721 as indicated. Western blots were used to analyze the expression of genes related to apoptosis induction. (C) Apoptosis induction in vivo. NSG mice harboring HuT78 tumors (n = 4 per group) were treated by gavage with a single dose of NT1721 (20 mg/kg) or the vehicle (3.3% DMSO/30% solutol in PBS). The tumors were then harvested at the indicated time points and subjected to Western blot analysis. Original Western Blots of Figure 3B,C available in Figure S5.

Figure 4

NT1721 suppressed tumor growth significantly better than romidepsin in a CTCL mouse model. (A) NSG mice (n = 7/group) were injected s.c. with HuT78 cells and treated by gavage three times/week (on consecutive days) with the vehicle control (30% solutol/3.3% DMSO in PBS), 20 mg/kg NT1721 or with 2 mg/kg romidepsin by I.P. injection twice per week. Treatment with NT1721 reduced tumor volumes significantly better than romidepsin on Day 4, 7 and 11 (p values < 0.01). (B) Weight of HuT78 tumors from individual mice (p = 0.0003 for control vs. NT1721 and for romidepsin vs. NT1721; no significant difference for control vs. romidepsin: p = 0.317). (C) Picture of HuT78 tumors from the treated mice. (D) NSG mice were injected s.c. with HH cells and treated by gavage on three consecutive days per week with 20 mg/kg NT1721 or the vehicle control. The differences in tumor volume were significant from day 14 through day 25 (p values < 0.0012). (E) Weight of HH tumors from individual mice (p = 0.0003). (F) Picture of representative HH tumors from the treated mice.

Figure 5

In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing 10% saline). (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor weight in treated and control mice. The differences between control and treatment groups as well as between the treatment groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721 once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week: p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002).

Figure 6

In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the two drugs for 48 h. (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to apoptosis iHut78 H. Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6.