NF-κB may be a potential therapeutic target for acute myelogenous leukemia (AML) because NF-κB activation is found in primitive human AML blast cells. In this report, we initially discovered that the potent antineoplastic effect of niclosamide, a Food and Drug Administration-approved antihelminthic agent, was through inhibition of the NF-κB pathway in AML cells. Niclosamide inhibited the transcription and DNA binding of NF-κB. It blocked tumor necrosis factor-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Niclosamide inhibited the steps TAK1→IκB kinase (IKK) and IKK→IκBα. Niclosamide also increased the levels of reactive oxygen species (ROS) in AML cells. Quenching ROS by the glutathione precursor N-acetylcysteine attenuated niclosamide-induced apoptosis. Our results together suggest that niclosamide inhibited the NF-κB pathway and increased ROS levels to induce apoptosis in AML cells. On translational study of the efficacy of niclosamide against AML, niclosamide killed progenitor/stem cells from AML patients but spared those from normal bone marrow. Niclosamide was synergistic with the frontline chemotherapeutic agents cytarabine, etoposide, and daunorubicin. It potently inhibited the growth of AML cells in vitro and in nude mice. Our results support further investigation of niclosamide in clinical trials of AML patients. Cancer Res; 70(6); 2516-27. ©2010 AACR.

Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of PRMT5 was observed in human CML LSCs. Silencing PRMT5 with shRNA or blocking PRMT5 methyltransferase activity with the small-molecule inhibitor PJ-68 reduced survival, serial replating capacity, and long-term culture-initiating cells (LTC-ICs) in LSCs from CML patients. Further, PRMT5 knockdown or PJ-68 treatment dramatically prolonged survival in a murine model of retroviral BCR-ABL-driven CML and impaired the in vivo self-renewal capacity of transplanted CML LSCs. PJ-68 also inhibited long-term engraftment of human CML CD34+ cells in immunodeficient mice. Moreover, inhibition of PRMT5 abrogated the Wnt/β-catenin pathway in CML CD34+ cells by depleting dishevelled homolog 3 (DVL3). This study suggests that epigenetic methylation modification on histone protein arginine residues is a regulatory mechanism to control self-renewal of LSCs and indicates that PRMT5 may represent a potential therapeutic target against LSCs.

Purpose: Resistance to STI571 is an emerging problem for patients with chronic myelogenous leukemia (CML). Mutation in the kinase domain of Bcr-Abl is the predominant mechanism of the acquired resistance to STI571. In the present study, we investigated the effect of triptolide on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. Experimental Design: CML cell lines (KBM5 versus KBM5-T315I, BaF3-Bcr-Abl versus BaF3-Bcr-Abl-T315I) and primary cells from CML patients with clinical resistance to STI571 were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. Nude mouse xenograft model was also used to evaluate the antitumor activity. Results: Triptolide potently down-regulated the mRNA and protein levels of Bcr-Abl independently of the caspase or proteosome activation in CML cells. It induced mitochondrial-dependent apoptosis in Bcr-Abl-T315I CML cells and primary cells from CML patients with clinical resistance to STI571. Additionally, triptolide inhibited the growth of STI571-sensitive KBM5 and STI571-resistant KBM5-T315I CML cells in nude mouse xenografts. Triptolide also down-regulated the expression of survivin, Mcl-1, and Akt in CML cells, which suggests that it may have multiple targets. Conclusions: These findings suggest that triptolide is a promising agent to overcome STI571-resistant CML cells, and warrant a clinical trial of triptolide derivatives for CML with Bcr-Abl-T315I mutation.

Summary The mesoderm- and epithelial-mesenchymal transition-associated transcription factor FOXC1 is specifically overexpressed in basal-like breast cancer (BLBC), but its biochemical function is not understood. Here we demonstrate that FOXC1 controls cancer stem cell (CSC) properties enriched in BLBC cells via activation of Smoothened (SMO)-independent Hedgehog (Hh) signaling. This non-canonical activation of Hh is specifically mediated by Gli2. We further show that the N-terminal domain of FOXC1 (aa 1–68) binds directly to an internal region (aa 898–1168) of Gli2, enhancing the DNA-binding and transcription-activating capacity of Gli2. FOXC1 expression correlates with that of Gli2 and its targets in human breast cancers. Moreover, FOXC1 overexpression reduces sensitivity to anti-Hedgehog (Hh) inhibitors in BLBC cells and xenograft tumors. Together, these findings reveal FOXC1-mediated non-canonical Hh signaling that determines the BLBC stem-like phenotype and anti-Hh sensitivity, supporting inhibition of FOXC1 pathways as potential approaches for improving BLBC treatment.

BackgroundChronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB) transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I.ResultsIn this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways.ConclusionTo our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for further drug development to overcome imatinib resistance in CML patients.