Yanli Jin

Triptolide Inhibits Bcr-Abl Transcription and Induces Apoptosis in STI571-resistant Chronic Myelogenous Leukemia Cells Harboring T315I Mutation

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.

The BH3-mimetic GX15-070 induces autophagy, potentiates the cytotoxicity of carboplatin and 5-fluorouracil in esophageal carcinoma cells

Despite improvements in both surgical techniques and radio- and chemo-therapy regimens, the prognosis of esophageal cancer is poor. In pursuit of novel effective strategy, this study examined the effect of the BH3-mimetic GX15-070 on esophageal carcinoma cells. We discovered that GX15-070 inhibited the growth of esophageal cancer cells. There was synergism between GX15-070 and carboplatin or 5-fluorouracil. GX15-070 induced autophagy in esophagus cancer cell line EC9706 and osteosarcoma cancer cell line U2OS. 3-methyladenine and chloroquine, inhibitors of autophagy with distinct mechanisms, potentiated the cytotoxicity of GX15-070. In conclusion, GX15-070 inhibits growth of esophageal cancer cells.

Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

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.

Differential impact of structurally different anti-diabetic drugs on proliferation and chemosensitivity of acute lymphoblastic leukemia cells

Hyperglycemia during hyper-CVAD chemotherapy is associated with poor outcomes of acute lymphoblastic leukemia (ALL) (Cancer 2004; 100:1179–85). The optimal clinical strategy to manage hyperglycemia during hyper-CVAD is unclear. To examine whether anti-diabetic pharmacotherapy can influence chemosensitivity of ALL cells, we examined the impacts of different anti-diabetic agents on ALL cell lines and patient samples. Pharmacologically achievable concentrations of insulin, aspart and glargine significantly increased the number of ALL cells, and aspart and glargine did so at lower concentrations than human insulin. In contrast, metformin and rosiglitazone significantly decreased the cell number. Human insulin and analogs activated AKT/mTOR signaling and stimulated ALL cell proliferation (as measured by flow cytometric methods), but metformin and rosiglitazone blocked AKT/mTOR signaling and inhibited proliferation. Metformin 500 μM and rosiglitazone 10 μM were found to sensitize Reh cells to daunorubicin, while aspart, glargine and human insulin (all at 1.25 mIU/L) enhanced chemoresistance. Metformin and rosiglitazone enhanced daunorubicin-induced apoptosis, while insulin, aspart and glargine antagonized daunorubicin-induced apoptosis. In addition, metformin increased etoposide-induced and L-asparaginase-induced apoptosis; rosiglitazone increased etoposide-induced and vincristine-induced apoptosis. In conclusion, our results suggest that use of insulins to control hyperglycemia in ALL patients may contribute to anthracycline chemoresistance, while metformin and thiazolidinediones may improve chemosensitivity to anthracycline as well as other chemotherapy drugs through their different impacts on AKT/mTOR signaling in leukemic cells. Our data suggest that the choice of anti-diabetic pharmacotherapy during chemotherapy may influence clinical outcomes in ALL.

Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia

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.

GDP366, a novel small molecule dual inhibitor of survivin and Op18, induces cell growth inhibition, cellular senescence and mitotic catastrophe in human cancer cells.

Accumulating evidence indicates that survivin plays a pivotal role in not only cell survival but also cell cycle progression. Op18/stathmin is an oncoprotein that regulates microtubule stabilization. Both survivin and Op18 have been proposed as therapeutic targets for cancer. However, few small molecule inhibitors of survivin and Op18 have been reported. In this study, we have identified a novel small molecule compound (GDP366) which potently and selectively inhibited the expression of both survivin and Op18. It decreased both the mRNA and protein levels of survivin and Op18. This inhibitory effect was not dependent on the status of p53 and p21 although GDP366 potently increased p53 and p21 levels. GDP366 significantly inhibited the growth of tumor cells in vitro and in vivo (nude mouse model) without rapid induction of apoptosis. GDP366 induced polyploidy in multiple types of cancer cell lines. GDP366 increased chromosomal instability, and induced cellular senescence by inhibiting telomerase activity. We conclude that GDP366 is a novel dual inhibitor of survivin and Op18. Our results warrant further translational evaluation of this compound.

Depletion of γ-catenin by Histone Deacetylase Inhibition Confers Elimination of CML Stem Cells in Combination with Imatinib.

Quiescent leukemia stem cells (LSCs) that are insensitive to BCR-ABL tyrosine kinase inhibitors confer resistance to imatinib in chronic myelogenous leukemia (CML). Identifying proteins to regulate survival and stemness of LSCs is urgently needed. Although histone deacetylase inhibitors (HDACis) can eliminate quiescent LSCs in CML, little is known about the underlying mechanism that HDACis kill LSCs. By fishing with a biotin-labeled probe, we identified that HDACi JSL-1 bound to the protein γ-catenin. γ-Catenin expression was higher in LSCs from CML patients than normal hematopoietic stem cells. Silencing γ-catenin in human CML CD34+ bone-marrow (BM) cells sufficiently eliminated LSCs, which suggests that γ-catenin is required for survival of CML LSCs. Pharmacological inhibition of γ-catenin thwarted survival and self-renewal of human CML CD34+ cells in vitro, and of murine LSCs in BCR-ABL-driven CML mice. γ-Catenin inhibition reduced long-term engraftment of human CML CD34+ cells in NOD.Cg-Prkdcscid II2rgtm1Sug/JicCrl (NOG) mice. Silencing γ-catenin by shRNA in human primary CD34+ cells did not alter β-catenin, implying a β-catenin-independent role of γ-catenin in survival and self-renewal of CML LSCs. Taken together, our findings validate that γ-catenin may be a novel therapeutic target of LSCs, and suppression of γ-catenin by HDACi may explain elimination of CML LSCs.

Tenovin-6-mediated inhibition of SIRT1/2 induces apoptosis in acute lymphoblastic leukemia (ALL) cells and eliminates ALL stem/progenitor cells

BackgroundAcute lymphoblastic leukemia (ALL) is a heterogeneous group of malignant disorders derived from B- or T-cell lymphoid progenitor cells. ALL often is refractory to or relapses after treatment; thus, novel targeted therapy for ALL is urgently needed. In the present study, we initially found that the level of SIRT1, a class III histone deacetylase, was higher in primary ALL cells from patients than in peripheral blood mononuclear cells from healthy individuals. But it is not clear whether inhibition of SIRT1 by its selective small molecule inhibitor Tenovin-6 is effective against ALL cells.MethodsWe tested the effect of Tenovin-6 on ALL cell lines (REH and NALM-6) and primary cells from 41 children with ALL and 2 adult patients with ALL. The effects of Tenovin-6 on cell viability were determined by MTS assay; colony-forming assays were determined by soft agar in ALL cell lines and methylcellulose medium in normal bone marrow cells and primary ALL blast cells; cell apoptosis and cell cycling were examined by flow cytometry; the signaling pathway was determined by Western blotting; ALL stem/progenitor cells were seperated by using MACS MicroBead kit.ResultsThe results showed that Tenovin-6 treatment activated p53, potently inhibited the growth of pre-B ALL cells and primary ALL cells, and sensitized ALL cells to frontline chemotherapeutic agents etoposide and cytarabine. Tenovin-6 induced apoptosis in REH and NALM-6 cells and primary ALL cells and diminished expression of Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP) in such cells. Furthermore, inhibition of SIRT1 by Tenovin-6 inhibited the Wnt/β-catenin signaling pathway and eliminated ALL stem/progenitor (CD133 + CD19-) cells.ConclusionOur results indicate that Tenovin-6 may be a promising targeted therapy for ALL and clinical trials are warranted to investigate its efficacy in ALL patients.

Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and β-catenin

BackgroundT674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib and dasatinib), similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.MethodsMolecular docking analysis in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα. The in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models.ResultsMolecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules (e.g., Stat3, Stat5). Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (IC50: 0.004–2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice.ConclusionsPonatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL.

The antihelminthic drug niclosamide effectively inhibits the malignant phenotypes of uveal melanoma in vitro and in vivo

Uveal melanoma (UM) is a lethal intraocular malignancy with an average survival of only 2~8 months in patients with hepatic metastasis. Currently, there is no effective therapy for metastatic UM. Here, we reported that niclosamide, an effective repellence of tapeworm that has been approved for use in patients for approximately 50 years, exhibited strong antitumor activity in UM cells in vitro and in vivo. We showed that niclosamide potently inhibited UM cell proliferation, induced apoptosis and reduced migration and invasion. p-Niclosamide, a water-soluble niclosamide, exerted potent in vivo antitumor activity in a UM xenograft mouse model. Mechanistically, niclosamide abrogated the activation of the NF-κB pathway induced by tumor necrosis factor α (TNFα) in UM cells, while niclosamide elevated the levels of intracellullar and mitochondrial reactive oxygen species (ROS) in UM cells. Quenching ROS by N-acetylcysteine (NAC) weakened the ability of niclosamide-mediated apoptosis. Matrix metalloproteinase 9 (MMP-9) knockdown by shRNA potentiated, while ectopic expression of MMP-9 rescued, the niclosamide-attenuated invasion, implying that MMP-9 is pivotal for invasion blockage by niclosamide in UM cells. Furthermore, our results showed that niclosamide eliminated cancer stem-like cells (CSCs) as reflected by a decrease in the Aldefluor+ percentage and serial re-plating melanosphere formation, and these phenotypes were associated with the suppressed Wnt/β-catenin pathway by niclosamide in UM. Niclosamide caused a dose- and time-dependent reduction of β-catenin and the key components [e.g., DVLs, phospho-GSK3β (S9), c-Myc and Cyclin D1] in the canonical Wnt/β-catenin pathway. Additionally, niclosamide treatment in UM cells reduced ATP and cAMP contents, and decreased PKA-dependent phosphorylation of β-catenin at S552 and S675 which determine the stability of β-catenin protein, suggesting that niclosamide may work as a mitochondrial un-coupler. Taken together, our results shed light on the mechanism of antitumor action of niclosamide and warrant clinical trial for treatment of UM patients.