c-Myc Levels Research Articles

Homoharringtonine Exhibits Anti-Tumor Effect in T-Cell Acute Lymphoblastic Leukemia By Targeting Notch1/Myc Pathway

T cell acute lymphoblastic leukemia (T-ALL) is a highly proliferative hematologic malignancy, mainly mediated by NOTCH/MYC signaling. Therefore, new therapies targeting NOTCH/MYC pathway are urgently required. Homoharringtonine (HHT), a potent inhibitor of translation elongation, has been used in China for many years for the treatment of myeloid leukemia and was approved in 2012 by the US Food and Drug Administration for the treatment of chronic myeloid leukemia with resistance to tyrosine kinase inhibitors. However, little is known about its efficacy in T-ALL. Herein, we examined the therapeutic role of HHT in T-ALL using mouse model and patient cells. First, we treated T-ALL cell lines (i.e., Jurkat, Molt4 and CCRF-CEM) and patient samples with HHT in vitro. After 24-hour(h) treatment, we observed very low IC50 values (5-20ng/mL) in all three T-ALL cell lines, suggesting highly sensitive to HHT treatment. HHT dramatically induced apoptosis and cell cycle arrest in G0/G1 phase in all three T-ALL cell lines. We observed similar results in human T-ALL cells treated with HHT for 24h, with much lower IC50 values (2-5ng/mL vs 30-110 ng/mL) in all three T-ALL patient samples compared to mononuclear cells from healthy donors, with increased apoptosis rates (89.2% vs 29.6%, p<0.001). Collectively, these results showed that T-ALL cell lines and patient samples exhibit high sensitivity to HHT in vitro. To evaluate the in vivo efficacy of HHT in T-ALL, we established a cohort of T-ALL mice by transplanting Luci+ Jurkat cells into NSG mice and then treated these mice with HHT (1mg/kg, ip) or vehicle (PBS) for 10 days, starting on day 9 post transplantation. Leukemia burden was compared by measuring Bioluminescence and engraftment at day 14 and day 19 post transplantation in HTT-treated and PBS-treated groups. We observed significantly decreased leukemia burden in HHT-treated mice compared to PBS-treated controls (median total flux: 796.3 vs 3701*106 at day 19, P=0.0001). HHT significantly prolonged the survival of the T-ALL mice as compared to PBS treatment (median survival: 29 vs 25 days, P=0.0003). T-ALL patient-derived-xenograft (PDX) model was also constructed and treated with HHT or PBS for 10 days. We observed significantly reduced human T-ALL engraftment rates (14.4-25.3% vs 37.8-65.3% at day 10 post transplantation, p<0.0001) and prolonged survival (median survival: 54 vs 45 days, P=0.0098) in the HHT-treated PDX compared to PBS-treated PDX, suggesting an in vivo anti-leukemia effect of HHT in T-ALL. Next, we performed RNA-seq of T-ALL cells with or without HHT treatment to identify downstream pathways that are regulated by HHT. Through gene set enrichment analysis (GSEA), we found that Myc pathway and Notch pathway were among the top 6 upregulated gene sets. Further, using western-blot analysis, we showed that all the expression levels of Notch1, c-MYC and MCL-1 were decreasing in T-ALL cell lines and patient samples after exposing to increased concentrations of HHT, suggesting that they were all down-regulated by HHT. Interestingly, we observed that a much lower concentration of HHT is needed to down-regulate Notch 1 (4ng/mL) than that needed to down-regulate MCL1(8-16ng/mL). Similar results were found in T-ALL human samples. Taken together, these results suggest that HHT could attenuate NOTCH 1 and MYC expression in T-ALL cells. Further, we used the Notch-1 induced T-ALL mouse model to verify the effect of HHT on Notch pathway and test its in vivo anti-ALL efficacy. Strikingly, we found that HHT treatment significantly reduced white blood cell (WBC) counts (0.82-1.68 *109/L vs 9.73-17.46*109/L, p<0.0001) and T-ALL burden (0.0-0.8% vs 7.9-57.1%, p=0.0003) and significantly prolonged survival (median survival: not reached vs 31 days, p=0.0007) as compared to PBS treatment. Interestingly, 60% (six of ten) HHT-treated T-ALL mice versus 0% of PBS-treated mice, obtained long-term survival, free of T-ALL after monitoring for 75 days, suggesting that these mice likely are cured by HHT monotherapy. QPCR and western-blot analysis using bone marrow cells of the treated mice showed that HHT dramatically reduced both the mRNA and protein levels of NOTCH1 compared to the control group. In summary, we showed here that HHT, approved by the FDA for CML treatment, exhibited in vivo therapeutic efficacy in T-ALL, by inhibiting the NOTCH-MYC pathway. Our data support to explore HHT as a novel therapy for T-ALL in the clinic.

Delineating CDK9- Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Abstract Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, and RNA Pol II, but not other genes (e.g., Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, and RNA Pol II, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 reduced mRNA/ protein levels of CDK9 as well as of Mcl-1, cMyc, RNA Pol II, and Mdm2, and increased p53 levels in a dose- and time-dependent manner, both in MM cell line and primary cells. In contrast, Thal-sns-032 did not inhibit survival of peripheral blood mononuclear cells (PBMCs) from healthy donors. Furthermore, Thal-sns-032 induced downregulation of CDK9, Mcl-1 and cMyc in MM.1S co-cultured with BMSCs cells. Of note, Thal-sns-032- mediated anti-MM effects were abrogated upon its wash-out. Moreover, knockdown CDK9 by shRNA inhibited proliferation and survival, both in MM tumor cell- and tumor cell/BMSC co-cultures. Rationally derived combination strategies of Thal-sns-032 with venetoclax, navitoclax, Selinexor or Carfilzomib as well as other investigational and established MM therapies induced synergistic anti-MM effects in MM cells or BMSC co-cultures. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derived MM combination treatment strategies.

HDAC Inhibition Involves CD26 Induction on Multiple Myeloma Cells Via the c-Myc/Sp1-Mediated Prompter Activation and Overcomes Therapeutic Resistance By Humanized Antibody

Background: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, little is known about its roles in plasma cell malignancies. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous due to its cellular diversity or immune escape, leading to a marked heterogeneity of response to humanized anti-CD26 monoclonal antibody (mAb) therapy in MM. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying innate and acquired resistance to CD26mAb therapy in MM. Aim: Here, we clarify the potential impact of epigenetic modification by HDAC inhibition (HDACi) with isoform selective- or broad- inhibitors on the regulation of CD26 expression in MM cells and elucidate its mechanisms, thereby affecting the performance of CD26mAb in refractory MM. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), cultured alone, the increased expression in CD26 levels of MM cells was detectable within 24 hr of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA by flow cytometry. Its expression increased further during the exposure to each HDACi and its maximum increase was observed at 72 hr of the treatment. Then, subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression on MM cells to reduced or baseline levels. In addition, the levels of CD26mRNA were concomitantly augmented, which was paralleled with an increase in the induction of CD26 protein and its DPPIV enzymatic activity in MM cells. We also examined the effect of each HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb.CD26mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining each HDACi plus CD26mAb at 10 μg/ml synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or CD26mAb alone. Next, gene expression profiles in KMS11, KMS27 and RPMI8226, left untreated or treated with LBH589 or RG2833 revealed that Myc is one of the overlapped genes, altered in its expression. The 5'-flanking region of human CD26 gene, representing the promoter activity contains potential binding sites of several transcriptional factors including Sp1 and c-Myc. So, further to elucidate the mechanisms by which CD26 expression is induced in MM cells by HDACi, we examined the epigenetic status in histone and non-histone at the CD26 promoter of MM cells with the treatment of HDACi. First, ChIP-qPCR demonstrated that the acetylation in histone 3 lysine 27 is increased at the CD26 promoter of 5 MM cell lines after exposure to each HDACi, suggesting parts of mechanisms in the CD26 promoter activation. Moreover, the expression level of c-Myc was time-dependently decreased in KMS11, KMS27 and RPMI8226 with the treatment of LBH-589 or RG2833 both at mRNA and protein levels and was significantly down-regulated after 12 to 24 hr of exposure. On the other hand, the acetylation of c-Myc on K323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the recovery % IP/INPUT showing the binding of c-Myc to the CD26 promoter was exposure time-dependently reduced in KMS11, KMS27 and RPMI8226 by the exposure to LBH589 or RG2833. Namely, in the absence of HDACi, c-Myc was present on the CD26 promoter via binding to Sp1 on the proximal G-C box and repressed CD26 promoter in MM cells, whereas, in the presence of HDACi, c-Myc was highly detached from Sp1 on the CD26 promoter after 12 to 24 hr with its increased acetylation, leading to activate CD26 promoter and initiate CD26 transcription in MM cells. Conclusion: HDACi not only shows anti-MM activity itself but sensitizes MM cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity.

Asparagine Bioavailability Regulates the Translation of MYC oncogene in Lymphoid Malignancies

Non-essential amino acids are becoming increasingly important in cancer metabolism because of their roles beyond protein translation. These include providing precursors for macromolecule biosynthesis, serving as substrates for epigenetic modifications, regulating redox states, and modulating cell signaling. Acute lymphoblastic leukemia (ALL) is one such example where asparagine is shown to be conditionally essential for cancer growth and proliferation. Exploiting this metabolic vulnerability, L-Asparaginase (ASNase) has been used in the clinic to successfully treat ALL patients through depleting the circulating asparagine. Our investigation into the mechanism of resistance to ASNase in ALL cells yielded a striking correlation between asparagine bioavailability and c-MYC protein levels. c-MYC is a key transcription factor and has been shown to regulate the transcription of more than 10% of genes, including many metabolic genes, to meet the demands of tumor growth and progression. In addition, it has a well-documented role in normal hematopoiesis and hematological malignancies, including ALL, where high c-MYC levels have been shown to be a marker for leukemic initiation and growth. Using asparagine auxotrophic ALL cells that do not express asparagine synthetase (ASNS), the asparagine biosynthesis enzyme, we found that c-MYC protein levels were acutely depleted when asparagine was withdrawn from the tissue culture medium. However, c-MYC protein levels were sustained when ASNS cDNA was re-introduced into these cells. These results indicate the possibility of a reciprocal association between metabolism and a critical oncogene MYC. We also discovered that asparagine depletion inhibited the translation of c-MYC without changing its mRNA expression or altering the rate of protein degradation. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and General Controlled Nonderepressible 2 (GCN2) are the two central molecular components involved in amino acid sensing and translation control. Asparagine starvation notably caused a decrease in global protein synthesis, mediated by suppression of mTORC1 and simultaneous activation of GCN2. Our studies using small molecule inhibitors and polysome profiling showed that repression of c-MYC mRNA translation is not due to the activation of the GCN2 pathway or a consequence of global translation suppression. In addition, both the '5' and '3' untranslated regions (UTRs) were not required for this inhibitory effect on c-MYC mRNA translation during asparagine depletion. Our results suggest that while asparagine depletion causes a defect in global translation at the initiation step, its effect on MYC mRNA translation is possibly due to elongation stalling. While ASNase can efficiently deplete circulating asparagine, its clinical applications are limited to ALL due to its low-level expression of ASNS. However, aspartate, a critical substrate for asparagine biosynthesis, can be pharmacologically targeted using electron transport chain (ETC) inhibitors. Our studies in ASNase-resistant ALL and lymphomas showed that while asparagine depletion alone did not affect c-MYC protein expression, co-treatment with ETC inhibitors (phenformin) showed a significant decrease in c-MYC protein levels. We extended this observation to test the synergy of ASNase and phenformin to treat an orthotopic MYC-driven B-cell lymphoma model. While either of the single treatments only modestly reduced the tumor burden, the double treatment showed the most significant reduction in the tumor burden, which correlated with a significant reduction of c-MYC protein expression. These results suggest that perturbing the asparagine biosynthesis pathway can be clinically employed to inhibit c-MYC protein expression and improve therapeutic outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aberrant expression of KDM1A inhibits ferroptosis of lung cancer cells through up-regulating c-Myc

Ferroptosis is a cell death process caused by metabolic dysfunction with the feature of aberrant iron accumulation. Emerging studies have identified that ferroptosis is an important biological function involving in the tumorigenesis, and targeting ferroptosis could provide promising therapeutic targets for lung cancer. However, such therapeutic strategies show limited therapeutic effect owing to drug resistance and other unknown underlying mechanisms. In this study, lysine-specific demethylase 1 (LSD1/KDM1A) was found to be significantly upregulated in lung cancer cells and tissues. The patients with KDM1A downregulation displayed the good prognosis. Using gene set enrichment analysis (GSEA), we demonstrated that KDM1A-associated genes might participate in the regulation of cell ferroptosis and Myc signaling in lung cancer. Knockdown of KDM1A inhibited the level of c-Myc and increased the concentration of malondialdehyde (MDA) and irons in human lung cancer cells H1299 and A549. Downregulation of c-Myc could facilitate KDM1A knockdown-mediated ferroptosis. Our study has elucidated the effect of KDM1A/c-Myc regulatory axis in the ferroptosis resistance of lung cancer cells.

Desmocollin-2 inhibits cell proliferation and promotes apoptosis in hepatocellular carcinoma via the ERK/c-MYC signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most common cancers around the world with a poor prognosis. The main reason for poor prognosis is early stage HCC is inconspicuous so it is difficult to detect and effective treatment strategies are lacking for advanced HCC. In this context, novel molecular targets are urgently needed for the diagnosis and therapy of HCC. In this study, we investigated the expression level, biological function, and relative mechanism of Desmocollin-2(DSC2) in HCC. DSC2 expression levels were decreased significantly in HCC cell lines SMMC-7721(7721), Huh7, HCC-LM3(LM3), and MHCC-97H(97H), especially in LM3 cells, compared with human liver cell line L02(L02). DSC2 overexpression in LM3 cells could inhibit the proliferation (in vitro and in vivo), colony formation, migration, and invasion abilities of HCC cells, and promote cell apoptosis, while DSC2 inhibition in 7721 cells performed the opposite effect. Consistent with these results, regulating DSC2 expression in 7721 and LM3 cells could affect the expression levels of apoptosis-related proteins (Bax, Bcl-2, c-Caspase-3, Caspase-3, Caspase-8, and Survivin) and cell cycle-related proteins (Cyclin D1, Cyclin B1, CDK1, and CDK2). Furthermore, DSC2 expression was significantly negatively correlated with the levels of p-ERK and c-MYC in both LM3 and 7721 cell lines. These findings confirmed that DSC2 overexpression could inhibit the proliferation, migration, and invasion abilities while promoting apoptosis of HCC cells via the ERK/c-MYC signaling pathway. In a conclusion, DSC2 was a tumor suppressor with low expression in liver cancer.

Identification of highly efficacious PROTACs targeting BRD4 against acute myeloid leukemia: Design, synthesis, and biological evaluations

The abnormal activation of BRD4 accelerates the progression of acute myeloid leukemia (AML), developing more precise therapeutics to intervene BRD4 promise to be an excellent opportunity to avoid current limitations of chemotherapy in clinic. Herein, a range of small-molecule PROTACs with the privileged 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one scaffold were rationally designed, which harbored different carbon or ethylenedioxy chains to degrade BRD4 mediated by the E3 ubiquitin ligase CRBN. Among them, the most potential B24 exhibited remarkable BRD4 degradation and excellent anti-proliferative activities in MV4-11 cells, with values of DC50 and IC50 for 0.75 nmol/L and 0.4 nmol/L, respectively, which were better than the BRD4 inhibitor (+)-JQ-1. Notably, this compound could time-dependently degrade the target protein in the BRD4-, CRBN-, and proteasome-dependent manner. Besides, B24 dramatically decreased the level of proto-oncogene c-Myc, and induced cell apoptosis by arresting the cell cycle in G0/G1 phase, down-regulating Bcl-2 and up-regulating Bax to amplify apoptotic effectors. This proof-of-concept study also highlighted the feasibility of BRD4-based PROTACs as a more powerful strategy against AML.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

An inhibitor of BRD4, GNE987, inhibits the growth of glioblastoma cells by targeting C-Myc and S100A16

PurposeAmong children, glioblastomas (GBMs) are a relatively common type of brain tumor. BRD4 expression was elevated in GBM and negatively correlated with the prognosis of glioma. We investigated the anti-GBM effects of a novel BRD4 inhibitor GNE987.MethodsWe evaluated the anti-tumor effect of GNE987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, the size of xenografts, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism.ResultsIn vitro experiments showed that GNE987 significantly degraded BRD4, inhibited the proliferation of GBM cells, blocked the cell cycle, and induced apoptosis. Similarly, in vivo experiments, GNE987 also inhibited GBM growth as seen from the size of xenografts and Ki67 immunohistochemical staining. Based on Western blotting, GNE987 can significantly reduce the protein level of C-Myc; meanwhile, we combined ChIP-seq with RNA-seq techniques to confirm that GNE987 downregulated the transcription of S100A16 by disturbing H3K27Ac. Furthermore, we validated that S100A16 is indispensable in GBM growth.ConclusionGNE987 may be effective against GBM that targets C-Myc expression and influences S100A16 transcription through downregulation of BRD4.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

The Effect of Aflatoxin B1 on Tumor-Related Genes and Phenotypic Characters of MCF7 and MCF10A Cells.

The fungal toxin aflatoxin B1 (AB1) and its reactive intermediate, aflatoxin B1-8, 9 epoxide, could cause liver cancer by inducing DNA adducts. AB1 exposure can induce changes in the expression of several cancer-related genes. In this study, the effect of AB1 exposure on breast cancer MCF7 and normal breast MCF10A cell lines at the phenotypic and epigenetic levels was investigated to evaluate its potential in increasing the risk of breast cancer development. We hypothesized that, even at low concentrations, AB1 can cause changes in the expression of important genes involved in four pathways, i.e., p53, cancer, cell cycle, and apoptosis. The transcriptomic levels of BRCA1, BRCA2, p53, HER1, HER2, cMyc, BCL2, MCL1, CCND1, WNT3A, MAPK1, MAPK3, DAPK1, Casp8, and Casp9 were determined in MCF7 and MCF10A cells. Our results illustrate that treating both cells with AB1 induced cytotoxicity and apoptosis with reduction in cell viability in a concentration-dependent manner. Additionally, AB1 reduced reactive oxygen species levels. Phenotypically, AB1 caused cell-cycle arrest at G1, hypertrophy, and increased cell migration rates. There were changes in the expression levels of several tumor-related genes, which are known to contribute to activating cancer pathways. The effects of AB1 on the phenotype and epigenetics of both MCF7 and MCF10A cells associated with cancer development observed in this study suggest that AB1 is a potential risk factor for developing breast cancer.

Garmultin-A Incites Apoptosis in CB3 Cells Through miR-17-5p by Attenuating Poly (ADP-Ribose) Polymerase-1.

BackgroundLeukemia accounts for a large number of deaths, worldwide, every year. Treating this ailment is always a challenging job. Recently, oncogenic miRNA leading to apoptosis are highly promising targets of many natural products. In this study, Garmultin-A (GA), isolated from the bark of Garcinia multiflora, was elucidated for its anti-leukemic effect in CB3 cells.MethodsThe effect of the compound on CB3 cell viability was detected by MTT assay and apoptosis by FITC Annexin V/PI and Hochest 33258 staining. The western blot analysis assessed the BAX, BCL2, cMYC, pERK, and PARP-1 protein levels. Autodock analysis predicted the ligand–protein interactions. q-RT-PCR quantified the miR-17-5p expression. Luciferase assay confirmed the interaction between PARP-1 and miR-17-5p.ResultsWe uncover that GA leads to apoptosis by inducing overexpression of miR-17-5p and significantly downregulate PARP-1 protein levels in CB3 cells. The overexpression of miR-17-5p promotes apoptosis, and the miR-17-5p antagomirs restore GA-triggered apoptosis. Notably, we disclose that PARP-1 is a direct target of miR-17-5p. Increased pro-apoptotic and reduced anti-apoptosis protein levels were also observed in GA-treated CB3 cells.ConclusionThese results provide critical insights that GA could induce apoptosis in CB3 cells through targeting miR-17-5p by attenuating PARP-1. Thus, GA could act as a novel therapeutic agent for erythroleukemia.

AML-147 C-MYC Targeting by Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Exerts Profound Cell Kill In Vitro and In Vivo in Acute Myeloid Leukemia and Lymphomas

The oncoprotein c-Myc governs epigenome and transcriptome and is deregulated in 70% of all human cancers. MYC is highly expressed in TP53 mutant or venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been met with success. PROTACs or cerebron E3 ligase modulators (CELMoDs) are attractive modalities to specifically target hitherto undruggable oncoproteins. We developed the first c-Myc degrader GT19630 (GT19715, the salt form of GT19630). We tested it in cell-free, cellular assays and in animal studies. GT19630 effectively degraded oncogenic c-Myc protein (IC50 = 1.5 nM) in HL-60 cells. C-Myc was effectively pulled down by biotinylated GT19630 in a cell-free, in vitro affinity purification assay; and a proteasome inhibitor ixazomib completely blocked c-Myc degradation. IC50 of GT19715 in HL-60 cells was 1.8 nM, being considerably lower than 40.2 nM, an IC50 of normal myeloid progenitors in CFU assay, suggesting a therapeutic window. GT19630 shares chemical properties with other CELMoDs and proteomic analyses revealed degradation of translation termination factor G1 to S phase transition proteins 1 (GSPT1), an important factor in LSC survival (Surka et al. Blood 2021). Indeed, GT19630 effectively degrades GSPT1 along with complete degradation of c-Myc in a xenograft model with HL-60 cells, and inhibits tumor growth at a dose as low as 0.3 mg/kg/bid. GT19630 had no effect on normal myeloid lineages in rats at 6 mg/kg. GT19715 eliminates circulating blasts and prolongs survival in the c-Myc-driven systemic Daudi leukemia/lymphoma model. Importantly, GT19715 induces cell killing independent of TP53 status, and baseline c-Myc protein levels significantly correlated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout or mutations of TP53 (R2 = 0.86, P = 0.02). We found that MV4;11 ven resistant (VR) cells demonstrated elevated protein levels of c-Myc, and GSPT1 and exhibited greater sensitivity to GT19715compared to ven-sensitive parental cells. Finally, GT19715 significantly reduced human CD45+ AML blasts compared to vehicle control in vivo in an AML PDX model. First results with the novel dual c-Myc/GSPT1 degrader GT19715 demonstrate promising preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development.

Circ_0000554 is identified as a cancer-promoting circRNA in colorectal cancer by regulating the miR-1205/LASP1 axis

BackgroundColorectal cancer (CRC) is a prevalent malignant tumor with poor prognosis. Circular RNAs (circRNAs) are key regulators in the progression of CRC. Our study aimed to disclose the role of circ_0000554 in CRC.MethodsThe expression of circ_0000554, miR-1205 and LIM and SH3 protein 1 (LASP1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, invasion and migration were monitored using cell counting kit-8 (CCK-8) assay, EdU assay, transwell assay and wound healing assay respectively. The protein levels of C-myc, matrix metallopeptidase 2 (MMP-2) and LASP1 were detected by western blot. Tumor formation assay in nude mice was conducted to explore the role of circ_0000554 in vivo. The association between miR-1205 and circ_0000554 or LASP1 was identified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay.Resultscirc_0000554 was upregulated in CRC tissues and cells, high circ_0000554 expression was significantly linked to shorter overall survival. Downregulation of circ_0000554 restrained cell growth and metastasis while promoted apoptosis in vitro, and suppressed tumorigenesis of CRC in vivo. Furthermore, mechanism study and rescue experiments confirmed miR-1205 could be sponged by circ_0000554 and its inhibitor reversed the inhibitory effect of circ_0000554 silencing on CRC progression. LASP1 was a target gene of miR-1205 and the upregulation of LASP1 overturned miR-1205-induced effects on CRC cells. Circ_0000554 could elevate LASP1 expression via interacting with miR-1205.

Ginsenoside Rb1 improves intestinal aging via regulating the expression of sirtuins in the intestinal epithelium and modulating the gut microbiota of mice.

Intestinal aging seriously affects the absorption of nutrients of the aged people. Ginsenoside Rb1 (GRb1) which has multiple functions on treating gastrointestinal disorders is one of the important ingredients from Ginseng, the famous herb in tradition Chinese medicine. However, it is still unclear if GRb1 could improve intestinal aging. To investigate the function and mechanism of GRb1 on improving intestinal aging, GRb1 was administrated to 104-week-old C57BL/6 mice for 6 weeks. The jejunum, colon and feces were collected for morphology, histology, gene expression and gut microbiota tests using H&E staining, X-gal staining, qPCR, Western blot, immunofluorescence staining, and 16S rDNA sequencing technologies. The numbers of cells reduced and the accumulation of senescent cells increased in the intestinal crypts of old mice, and administration of GRb1 could reverse them. The protein levels of CLDN 2, 3, 7, and 15 were all decreased in the jejunum of old mice, and administration of GRb1 could significantly increase them. The expression levels of Tert, Lgr5, mKi67, and c-Myc were all significantly reduced in the small intestines of old mice, and GRb1 significantly increased them at transcriptional or posttranscriptional levels. The protein levels of SIRT1, SIRT3, and SIRT6 were all reduced in the jejunum of old mice, and GRb1 could increase the protein levels of them. The 16S rDNA sequencing results demonstrated the dysbiosis of the gut microbiota of old mice, and GRb1 changed the composition and functions of the gut microbiota in the old mice. In conclusion, GRb1 could improve the intestinal aging via regulating the expression of Sirtuins family and modulating the gut microbiota in the aged mice.

Melatonin inhibits ESCC tumor growth by mitigating the HDAC7/β-catenin/c-Myc positive feedback loop and suppressing the USP10-maintained HDAC7 protein stability.

BackgroundMelatonin, a natural hormone secreted by the pineal gland, has been reported to exhibit antitumor properties through diverse mechanisms of action. However, the oncostatic function of melatonin on esophageal squamous cell carcinoma (ESCC) remains elusive. This study was conducted to investigate the potential effect and underlying molecular mechanism of melatonin as single anticancer agent against ESCC cells.MethodsESCC cell lines treated with or without melatonin were used in this study. In vitro colony formation and EdU incorporation assays, and nude mice tumor xenograft model were used to confirm the proliferative capacities of ESCC cells. RNA-seq, qPCR, Western blotting, recombinant lentivirus-mediated target gene overexpression or knockdown, plasmids transfection and co-IP were applied to investigate the underlying molecular mechanism by which melatonin inhibited ESCC cell growth. IHC staining on ESCC tissue microarray and further survival analyses were performed to explore the relationship between target genes’ expression and prognosis of ESCC.ResultsMelatonin treatment dose-dependently inhibited the proliferative ability and the expression of histone deacetylase 7 (HDAC7), c-Myc and ubiquitin-specific peptidase 10 (USP10) in ESCC cells (P < 0.05). The expressions of HDAC7, c-Myc and USP10 in tumors were detected significantly higher than the paired normal tissues from 148 ESCC patients (P < 0.001). Then, the Kaplan–Meier survival analyses suggested that ESCC patients with high HDAC7, c-Myc or USP10 levels predicted worse overall survival (Log-rank P < 0.001). Co-IP and Western blotting analyses further revealed that HDAC7 physically deacetylated and activated β-catenin thus promoting downstream target c-Myc gene transcription. Notably, our mechanistic study validated that HDAC7/β-catenin/c-Myc could form the positive feedback loop to enhance ESCC cell growth, and USP10 could deubiquitinate and stabilize HDAC7 protein in the ESCC cells. Additionally, we verified that inhibition of the HDAC7/β-catenin/c-Myc axis and USP10/HDAC7 pathway mediated the anti-proliferative action of melatonin on ESCC cells.ConclusionsOur findings elucidate that melatonin mitigates the HDAC7/β-catenin/c-Myc positive feedback loop and inhibits the USP10-maintained HDAC7 protein stability thus suppressing ESCC cell growth, and provides the reference for identifying biomarkers and therapeutic targets for ESCC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40779-022-00412-0.

Gene expression analysis and the risk of relapse in favorable histology Wilms’ tumor

ABSTRACT Introduction and Objectives Wilms’ tumor (WT) relapse occurs in 15% of patients. We aim to investigate the association between the expression of several genetic markers and WT relapse risk. Materials and methods The study included 51 children treated for WT at a tertiary center between 2001 and 2019: 23 patients had disease relapse (group A) and 28 remained relapse-free after at least 2 years of follow-up (group B). Patients with syndromic, bilateral synchronous or anaplastic WT were excluded. Autologous renal tissue from 20 patients served as control. Total RNA was isolated from tumor tissue and control. Gene expression levels of WT1, HIF1α, b-FGF, c-MYC and SLC22A18 were assessed using quantitative RT-PCR and normalized to GAPDH. Immunohistochemical staining for WT1 and gene expression levels were compared between the study groups. Results Median patient age was 3 (IQR = 2–5) years and 36 (70.6%) had stage I disease. Baseline characteristics were similar between study groups. Relapse occurred at a median of 6.8 (2.8–24.7) months, predominantly in the lungs (11/23, 47.8%). Tumors that relapsed expressed significantly higher levels of WT1, HIF1α, b-FGF and c-MYC and lower levels of SLC22A18 (p < 0.001). Strong immunohistochemical staining for WT1 was seen in 73.9% of group A and 14.29% of group B (p < 0.001). These associations retained statistical significance irrespective of patient and tumor characteristics. Conclusions Higher expression levels of WT1, HIF1 α, b-FGF and c-MYC and lower level of SLC22A18 are associated with increased risk of WT relapse. These genetic markers can serve as future prognostic predictors and help stratify patients for treatment.

C-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

FUT6 inhibits the proliferation, migration, invasion, and EGF-induced EMT of head and neck squamous cell carcinoma (HNSCC) by regulating EGFR/ERK/STAT signaling pathway.

Glycosylation change is one of the landmark events of tumor occurrence and development, and tumor cells may be inhibited by regulating the aberrant expression of glycosyltransferases. Currently, fucosyltransferase VI (FUT6), which is involved in the synthesis of α-1, 3 fucosyl bond, has been detected to be closely associated with multiple tumors, but its function and mechanism in head and neck squamous cell carcinoma (HNSCC) still need further research. In this study, FUT6 knockdown and overexpression strategies were used to investigate the effects of FUT6 on cell proliferation, migration, and invasion, as well as the growth and metastasis of HNSCC in a xenografts mouse model. The protein expression levels of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Signal Transducer and Activator of Transcription (STAT), protein kinase B (AKT), c-Myc, and epithelial-mesenchymal transition (EMT) markers were determined by western blot analysis. Our research found that the mRNA expression of FUT6 was lower in HNSCC tissues than in normal mucosal epithelial tissues. In Cal-27 and FaDu cells, FUT6 overexpression inhibited cell proliferation, migration and invasion, causing upregulation of ZO-1 and E-cadherin, downregulation of N-cadherin and Vimentin, and finally decreased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In HSC-3 cells, knockdown of FUT6 promoted cell proliferation, migration and invasion, downregulating ZO-1 and E-cadherin, upregulating N-cadherin and Vimentin, and increased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In the HNSCC xenografts mouse, FUT6 overexpression inhibited tumor growth and metastasis. In summary, FUT6 controls the proliferation, migration, invasion, and EGF-induced EMT of HNSCC by regulating EGFR/ERK/STAT signaling pathway, indicating its potential future therapeutic application for HNSCC.

Termination of CD40L co-stimulation promotes human B cell differentiation into antibody-secreting cells.

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (TFH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and TFH cells are transient; after TFH contact, B cells cycle between GC light and dark zones where TFH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs.