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PDF file - 185KB, The effect of Ponatinib on beta-catenin signaling.

PDF file - 134KB, The analysis of KIT mutation.

Supplementary Data from Sustained c-Jun-NH2-Kinase Activity Promotes Epithelial-Mesenchymal Transition, Invasion, and Survival of Breast Cancer Cells by Regulating Extracellular Signal-Regulated Kinase Activation

PDF file - 43KB, The primers are listed.

PDF file - 158KB, Peripheral blood cell counts of ASM/MCL mice.

PDF file - 137KB, Immunohistochemical analysis of the in vivo signaling in xenograft tumors.

Gain-of-function mutations of membrane receptor tyrosine kinase KIT, especially gatekeeper D816V point mutation in KIT, render kinase autoactivation, disease progression, and poor prognosis. D816V KIT is found in approximately 80% of the patients with systemic mastocytosis, and is resistant to the first and second generations of tyrosine kinase inhibitors (TKI). The purpose of this investigation was aimed at exploring whether ponatinib (AP24534), a novel effective TKI against T315I Bcr-Abl, was active against D816V KIT. We discovered that ponatinib abrogated the phosphorylation of KIT harboring either V560G (sensitive to imatinib) or D816V mutation (resistant to imatinib) and the downstream signaling transduction. Ponatinib inhibited the growth of D816V KIT–expressing cells in culture and nude mouse xenografted tumor. Ponatinib triggered apoptosis by inducing the release of cytochrome c and AIF, downregulation of Mcl-1. Furthermore, ponatinib abrogated the phosphorylation of β-catenin at the site Y654, suppressed the translocation of β-catenin, and inhibited the transcription and DNA binding of TCF and the expression of its targets (e.g., AXIN2, c-MYC, and CCND1). Moreover, ponatinib was highly active against xenografted D816V KIT tumors in nude mice and significantly prolonged the survival of mice with aggressive systemic mastocytosis or mast cell leukemia by impeding the expansion and infiltration of mast cells with imatinib-resistant D814Y KIT. Our findings warrant a clinical trial of ponatinib in patients with systemic mastocytosis harboring D816V KIT. Mol Cancer Ther; 13(5); 1217–30. ©2014 AACR.

The c-Jun NH2-terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2–mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs. Mol Cancer Res; 8(2); 266–77

AXL knockdown has minimal effect on self-renewal capacity of human NBM CD34+ cells.

Supplementary Information

Supplementary Figure 2 from The Antitumor Activity of Homoharringtonine against Human Mast Cells Harboring the KIT D816V Mutation

PCR Primers

Niclosamide promotes proteosome-dependent degradation of β-catenin and abrogates nuclear translocation of FOXM1 and β-catenin in CML cells.

PDF file - 187K

Figure S1 - p53-Null K562 and KCL-22 cells were resistance to MLN4924; Figure S2 - MLN4924 induces apoptosis in IM-sensitive and -resistant CML cells harboring WT-p53 in a time-dependent fashion; Figure S3 - MLN4924 suppresses survival of BCR-ABL+ CML LSCs in vivo; Figure S4 - p27kip1 knockdown attenuates the effect of MLN4924 on CML LSCs in vivo.

Table S1 shows information of antibodies for Western blotting analysis.

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

Table S1 shows genetic status of the known altered genes in UM cell lines; Table S2 shows detailed information for 45 patients with UM; Table S3 shows the association between NAE1 expression and clinicopathologic features in UM patients; Table S4 shows limiting dilution analysis in NOD-SCID mice; Table S5 shows primers for qRT-PCR analysis.

Purpose: Liver metastasis is the major and direct cause of death in patients with uveal melanoma (UM). There is no effective therapy for patients with metastatic UM. Improved treatments of hepatic metastatic patients with UM were urgently needed. Inspired by readily detectable key components in the neddylation pathway in UM cells, we aimed at exploring whether neddylation pathway was a therapeutic target for liver metastatic UM.Experimental Design: Expression of key proteins in the neddylation pathway in UM was detected by Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunohistochemical staining. Cellular proliferation, apoptosis, cell cycle, migration, and cancer stem-like cells (CSCs) properties were examined upon treatment with MLN4924, a potent and selective NAE inhibitor. Antitumor activity and frequency of CSCs were determined by using a NOD-SCID mouse xenograft model. Liver metastasis was evaluated by use of a NOD-scid-IL2Rg−/− mouse model.Results: NAE1 expression was readily detectable in UM. Inhibition of the neddylation pathway by MLN4924 repressed the CSCs properties in UM (capacities of tumorsphere formation and serially replating, aldehyde dehydrogenase-positive cells, and frequency of CSC) through Slug protein degradation. MLN4924 treatment disturbed the paracrine secretion of NF-κB-mediated VEGF-C and its dependent angiogenesis. The inhibitory effect of neddylation blockade on proliferation, which was confirmed by xenografted UM tumor in NOD-SCID mice, was involved in activation of ATM-Chk1-Cdc25C DNA damage response, and G2–M phase arrest. Neddylation inhibition profoundly inhibited hepatic metastasis in UM.Conclusions: Our studies validate the neddylation pathway as a promising therapeutic target for the treatment of patients with hepatic metastasis of UM. Clin Cancer Res; 24(15); 3741–54. ©2017 AACR.See related commentary by Yang et al., p. 3477

Combination of niclosamide and imatinib eliminated LSCs in vitro.

Purpose: The “gate-keeper” mutations T674I platelet—derived growth factor receptor α (PDGFRα) in hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to efficiently kill resistant malignant hematologic cells. The purpose of this study was to evaluate the strategy of shutting down the transcription and expression of FIP1-like-1 (FIP1L1)–PDGFRα and Bcr-Abl with SNS-032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials.Experimental Design: The effects of SNS-032 on PDGFRα and Bcr-Abl signaling pathways, apoptosis, and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRα and KBM5-T315I Bcr-Abl cells in nude mouse models.Results: SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032 decreased both the mRNA and protein levels of FIP1L1-PDGFRα and Bcr-Abl and inhibited the proliferation of malignant cells expressing FIP1L1-PDGFRα or Bcr-Abl. It also decreased the phosphorylation of downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death receptor pathway.Conclusions: This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRα–positive HES cells and Bcr-Abl–positive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating hematologic malignancy by abrogating oncogene addiction. Clin Cancer Res; 18(7); 1966–78. ©2012 AACR.

Supplementary Materials，Methods and Figure Legends

Purpose: Quiescent leukemia stem cells (LSC) are important resources of resistance and relapse in chronic myelogenous leukemia (CML). Thus, strategies eradicating CML LSCs are required for cure. In this study, we discovered that AXL tyrosine kinase was selectively overexpressed in primary CML CD34+ cells. However, the role of AXL and its ligand Gas6 secreted by stromal cells in the regulation of self-renewal capacity of LSCs has not been well investigated.Experimental Design: The function of CML CD34+ cells was evaluated by flow cytometer, CFC/replating, long-term culture-initiating cells (LTC-IC), CML mouse model driven by human BCR-ABL gene and NOD-scid-IL2Rg−/− (NSI) mice.Results: AXL was selectively overexpressed in primary CML CD34+ cells. AXL knockdown reduced the survival and self-renewal capacity of human CML CD34+ cells. Pharmacologic inhibition of AXL reduced the survival and self-renewal capacity of human CML LSCs in vitro and in long-term grafts in NSI mice. Human CML CD34+ cells conscripted bone marrow–derived stromal cells (BMDSC) and primary mesenchymal stem cells (MSC) to secrete Gas6 to form a paracrine loop that promoted self-renewal of LSCs. Suppression of AXL by shRNA and inhibitor prolonged survival of CML mice and reduced the growth of LSCs in mice. Gas6/AXL ligation stabilizes β-catenin in an AKT-dependent fashion in human CML CD34+ cells.Conclusions: Our findings improve the understanding of LSC regulation and validate Gas6/AXL as a pair of therapeutic targets to eliminate CML LSCs. Clin Cancer Res; 23(11); 2842–55. ©2016 AACR.

Supplementary Information

Figure S1 - Biological and pharmacological inhibition of EZH2 potently dampens growth of IM-sensitive and -resistant CML cells; Figure S2 - GSK126 induces apoptosis in CML cells in a dose- and time-dependent manner; Figure S3 - GSK126 elicits cytochrome c release and mitochondrial damage; Figure S4 - Administration of GSK126 in CML mice reduces LSCs in spleen nucleated cells from CML mice; Figure S5 - Knockdown of EZH2 reduces in vivo LSCs in CML mice; Figure S6 - Pharmacological and biological inhibition of EZH2 in CML mice has minimal effect on LSCs and progenitor cells in BM and spleen nucleated cells from CML mice; Figure S7 - BCR-ABL is not involved in the EZH2 knockdown-enabled PTEN transcriptional elevation; Figure S8 - Knockdown of PTEN weakens the effect of LSCs reduction mediated by silencing EZH2.

Supplementary Figure Legends 1-2 from The Antitumor Activity of Homoharringtonine against Human Mast Cells Harboring the KIT D816V 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.

Supplementary information

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G2–M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34+ cells and LSCs in CML-bearing mice via accumulation of p27kip1 in the nucleus. Notably, p27kip1 silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML.Significance: These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. Cancer Res; 78(6); 1522–36. ©2018 AACR.

Purpose: Leukemia stem cells (LSC), which are insensitive to tyrosine kinase inhibitors (TKI), are an important source of TKI resistance and disease relapse in chronic myelogenous leukemia (CML). Obstacles to eradicating LSCs include limited understanding of the regulation network of LSCs. The current study aimed to examine the interplay between NF-κB and FOXM1/β-catenin, and the effect of its chemical intervention on CML LSCs.Experimental Design: The interplay between NF-κB and FOXM1/β-catenin was analyzed by reciprocal coimmunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assay in CML cells. The effect of disturbing NF-κB and FOXM1/β-catenin by niclosamide on the self-renewal capacity and survival of LSCs was evaluated in vitro in human primary CML CD34+ cells and in vivo in CML mice.Results: Reciprocal co-IP experiments showed physical interaction of p65 and FOXM1. p65 promoted transcription of FOXM1 gene. ChIP assay revealed recruitment of p65 on the promoter of FOXM1 gene. Conversely, FOXM1 and β-catenin positively regulated the nuclear translocation and transcriptional activity of NF-κB in CML cells. Niclosamide disrupted the positive feedback loop between NF-κB and FOXM1/β-catenin, thereby impairing the self-renewal capacity and survival of CML LSCs. Niclosamide decreased the long-term engraftment of human CML LSCs in NOD-SCID IL2Rγ chain-deficient (NOG) mice, and prolonged the survival of CML mice.Conclusions: Interaction of p65 with FOXM1/β-catenin is critical in CML and its disruption by niclosamide eradicates LSCs. These findings may improve the understanding of a self-renewal regulatory mechanism of LSCs and offer a rationale-based approach to eliminate LSCs in CML. Clin Cancer Res; 23(3); 789–803. ©2016 AACR.

Table S1 shows the Characteristics of patients with CML; Table S2 shows the sequence of shRNA; Table S3 shows the limiting dilution assay of mouse LT-HSC after shEZH2 treatment

Purpose: Leukemia stem cells (LSCs) are an important source of tyrosine kinase inhibitor resistance and disease relapse in patients with chronic myelogenous leukemia (CML). Targeting LSCs may be an attractive strategy to override this thorny problem. Given that EZH2 was overexpressed in primary CML CD34+ cells, our purpose in this study was to evaluate the effects of targeting EZH2 on CML LSCs and clarify its underlying mechanism.Experimental Design: Human primary CML CD34+ cells and retrovirally BCR–ABL-driven CML mouse models were employed to evaluate the effects of suppression of EZH2 by GSK126- or EZH2-specific shRNA in vitro and in vivo. Recruitment of EZH2 and H3K27me3 on the promoter of tumor-suppressor gene PTEN in CML cells was measured by chromatin immunoprecipitation assay.Results: Our results showed that pharmacologic inhibition of EZH2 by GSK126 not only elicited apoptosis and restricted cell growth in CML bulk leukemia cells, but also decreased LSCs in CML CD34+ cells while sparing those from normal bone marrow CD34+ cells. Suppression of EZH2 by GSK126 or specific shRNA prolonged survival of CML mice and reduced the number of LSCs in mice. EZH2 knockdown resulted in elevation of PTEN and led to impaired recruitment of EZH2 and H3K27me3 on the promoter of PTEN gene. The effect of EZH2 knockdown in the CML mice was at least partially reversed by PTEN knockdown.Conclusions: These findings improve the understanding of the epigenetic regulation of stemness in CML LSCs and warrant clinical trial of GSK126 in refractory patients with CML. Clin Cancer Res; 24(1); 145–57. ©2017 AACR.

Supplementary Figure 1 from The Antitumor Activity of Homoharringtonine against Human Mast Cells Harboring the KIT D816V Mutation

Figure S1-The specificity of NAE1 antibody for immunohistochemical analysis was verified; Figure S2-MLN4924 induces apoptosis in human UM cells; Figure S3-MLN4924 is synergistic with vinblastine to induce apoptosis in UM cells; Figure S4-MLN4924 induces DNA damage response and G2/M-phase arrest in UM cells; Figure S5-Inhibiting checkpoint of DNA damage potentiates the cytotoxicity of MLN4924; Figure S6-MLN4924 suppresses ALDH+ cells in UM cells; Figure S7-Slug is required for maintenance of CSCs properties in UM cells; Figure S8-MLN4924 inhibits migration and invasion of human UM cells; Figure S9-MLN4924 inhibits hepatic metastasis of UM cells by attenuating VEGF-C secretion.