c-Myc Translation Research Articles

Depletion of NEAT1 lncRNA attenuates nucleolar stress by releasing sequestered P54nrb and PSF to facilitate c-Myc translation.

Altered expression of NEAT1, the architectural long non-coding RNA (lncRNA) of nuclear paraspeckles, has been reported during tumorigenesis, as well as under various cellular stress conditions. Here we report that the depletion of NEAT1 lncRNA alleviates nucleolar stress during RNAP I inhibition through releasing sequestered P54nrb and PSF to facilitate the IRES-dependent translation of c-Myc. RNAP I inhibitor CX5461 disrupts the SL1-rDNA interaction and induces nucleolar disruption, demonstrated by the accumulation of fibrillarin-containing nucleoplasmic foci and nucleolar clearance of ribosomal proteins in HeLa cells. Antisense oligonucleotide-mediated depletion of NEAT1 lncRNA significantly attenuated the RNAP I inhibition and its related nucleolar disruption. Interestingly, induction in the levels of c-Myc protein was observed in NEAT1-depeleted cells under RNAP I inhibition. NEAT1-associated paraspeckle proteins P54nrb and PSF have been reported as positive regulators of c-Myc translation through interaction with c-Myc IRES. Indeed, an increased association of P54nrb and PSF with c-Myc mRNA was observed in NEAT1-depleted cells. Moreover, apoptosis was observed in HeLa cells depleted of P54nrb and PSF, further confirming the positive involvement of P54nrb and PSF in cell proliferation. Together, our results suggest that NEAT1 depletion rescues CX5461-induced nucleolar stress through facilitating c-Myc translation by relocating P54nrb/PSF from nuclear paraspeckles to c-Myc mRNAs.

Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

Rhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults. Rhabdomyosarcomas are skeletal muscle-like tumours that typically arise in muscle beds, and express key myogenic regulatory factors. However, their developmental program remains blocked in the proliferative phase with cells unable to exit the cell cycle to fuse into myotubes. Recently, we uncovered a key role for the RNA-binding protein Staufen1 during myogenic differentiation through the regulation of c-myc translation. Given the known implication of c-myc in rhabdomyosarcoma, we hypothesized in the current work that Staufen1 controls rhabdomyosarcoma tumorigenesis. Here, we report for the first time the novel role of Staufen1 in cancer, specifically in rhabdomyosarcoma. We demonstrate that Staufen1 is markedly upregulated in human rhabdomyosarcoma tumours and cell lines as compared to normal skeletal muscle. Moreover, we show that Staufen1 promotes the tumorigenesis of embryonal and alveolar rhabdomyosarcoma subtypes both in cell culture and in animal models. Finally, our data demonstrate that Staufen1 has differential roles in embryonal versus alveolar rhabdomyosarcoma through the control of proliferative and apoptotic pathways, respectively. Together, these results provide the first evidence for Staufen1’s direct implication in cancer biology. Accordingly, Staufen1 thus represents a novel target for the development of future therapeutic strategies for rhabdomyosarcoma.

PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1.

The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator.

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies

AbstractPhosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc–dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.

Downregulation of p68 RNA Helicase (DDX5) Activates a Survival Pathway Involving mTOR and MDM2 Signals.

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5' end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translation.

Downregulation of p68 RNA Helicase (DDX5) Activates a Survival Pathway Involving mTOR and MDM2 Signals

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/ MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5’ end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translation.

Silencing c-Myc Translation As a Therapeutic Strategy through Targeting PI3K Delta and CK1 Epsilon in Hematological Malignancies

Introduction: c-Myc is a master transcription factor and one of the most frequently altered genes across a vast array of human cancers including diffuse large B-cell lymphoma (DLBCL), and is thus an attractive therapeutic target . However, no direct inhibitor of c-Myc has been successfully developed for the treatment of any cancer. The c-Myc protein has a short half-life of less than 30 minutes , and the complex secondary structures in the 5' untranslated region (UTR) of MYC mRNA make its translation highly dependent on the eukaryotic translation initiation factor 4F (eIF4F) . eIF4F exists as a complex comprised of the eIF4E, eIF4A, and eIF4G subunits. eIF4E can be sequestered by 4E-BP1, which acts as a "brake" for initiation of mRNA translation . Hyper-phosphorylation of 4E-BP1, caused by upstream signals such as mTORC1, leads to release of eIF4E from 4E-BP1, assembly of the eIF4F complex, and robust mRNA translation. Surprisingly, neither FDA approved mTORC1 inhibitors nor the investigational mTORC1/mTORC2 inhibitor MLN0128 has demonstrated adequate activity in aggressive lymphoma. The therapeutic effects of mTOR inhibition in c-Myc driven aggressive lymphoma remain poorly understood.Recognizing phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating mTOR, we hypothesized that co-targeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. Since both PI3K and proteasome are proven drug targets in blood cancer, such co-targeting strategy may be expeditiously studied in clinical trials for c-Myc driven aggressive lymphoma.Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo (Promega®). The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups. Mechanisms of synergy were determined through interrogating the effects of small molecule inhibitors and shRNA targeting regulators of various regulators of 4E-BP1. Structural studies of TGR-1202 were performed by in silico docking, and validated by synthesis of novel analogs of TGR-1202. Activity of TGR-1202 on CK1 epsilon was studied by kinome profiling (Reaction Biology®), cell free kinase assay of CK1e (Promega®), and cell based assay of CK1e autophosphorylation.Results: We found that a novel PI3K delta isoform inhibitor TGR-1202, but not the approved PI3Kd inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells (Figure 1). TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of eIF4E-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc dependent transcription (Figure 2). Furthermore, the synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kd inhibitors, was active against casein kinase-1 (CK1) epsilon (Figure 3). Targeting CK1e using a selective chemical inhibitor or shRNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc.Conclusion: These results suggest that TGR-1202 is a first-in-class dual PI3Kd/CK1e inhibitor, which may in part explain the preliminary clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1e should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. [Display omitted] [Display omitted] [Display omitted] DisclosuresLentzsch:BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria. O'Connor:Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Spectrum: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D’Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay (’Click-iT’ assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab’2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. DisclosuresSteele:Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

Quambalarine B, a Secondary Metabolite from Quambalaria cyanescens with Potential Anticancer Properties.

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.

Icariside II, a natural mTOR inhibitor, disrupts aberrant energy homeostasis via suppressing mTORC1-4E-BP1 axis in sarcoma cells.

The aberrant energy homeostasis that characterized by high rate of energy production (glycolysis) and energy consumption (mRNA translation) is associated with the development of cancer. As mammalian target of rapamycin (mTOR) is a critical regulator of aberrant energy homeostasis, it is an attractive target for anti-tumor intervention. The flavonoid compound Icariside II (IS) is a natural mTOR inhibitor derived from Epimedium. Koreanum. Herein, we evaluate the effect of IS on aberrant energy homeostasis. The reduction of glycolysis and mRNA translation in U2OS (osteosarcoma), S180 (fibrosarcoma) and SW1535 (chondrosarcoma) cells observed in our study, indicate that, IS inhibits aberrant energy homeostasis. This inhibition is found to be due to suppression of mammalian target of rapamycin complex 1 (mTORC1)-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) axis through blocking the assembly of mTORC1. Furthermore, IS inhibits the cap-dependent translation of c-myc through mTORC1-4E-BP1 axis which links the relationship between mRNA translation and glycolysis. Inhibition of aberrant energy homeostasis by IS, contributes to its in vitro and in vivo anti-proliferation activity. These data indicate that IS disrupts aberrant energy homeostasis of sarcoma cells through suppression of mTORC1-4E-BP1 axis, providing a novel mechanism of IS to inhibit cell proliferation in sarcoma cells.

Abstract B01: MicroRNAs as novel therapeutic adjuvants to treat high-risk medulloblastoma.

Abstract Medulloblastoma is the most common malignant brain tumor in children, accounting for 18% of newly diagnosed brain tumors and 10% of all children cancer-related deaths. Despite improvement in the 5-year survival rate of medulloblastoma in recent years, only a small percentage of patients survive high-risk metastatic disease. The quality of life for those who do survive is often substantially reduced due to the toxicity associated with radiation and chemotherapy. Vincristine is a microtubule-destabilizing antimitotic drug, which is routinely administered in higher dosages to both high and average risk medulloblastoma patients. As a result, these patients suffer from devastating neurotoxic effects that include but not limited to: sensorimotor and autonomic neuropathy, hearing loss, mononeuropathy, and seizures. Using high-throughput microRNA mimic library screens, we identified a group of microRNAs that may improve the efficacy of vincristine against c-MYC amplified medulloblastoma as well as re-sensitize vincristine-resistant medulloblastoma. Our findings revealed that these microRNAs may act as tumor suppressors since their overexpression inhibited colony formation, migration and invasion ability of medulloblastoma cells. Furthermore, these microRNAs suppressed stem cell renewal/proliferation of c-MYC amplified medulloblastoma cells. Expression analysis, gene enrichment analysis and target prediction algorithms revealed that these microRNAs exert their vincristine sensitizing and tumor suppressor effect by targeting genes involved in microtubule organization, cell cycle regulation, DNA damage repair and mRNA translation. One of our most interesting targets is EIF4E3, which is a translation initiation factor. Our preliminary findings indicate that EIF4E3 may regulate medulloblastoma cell growth, progression and vincristine sensitivity by modulating c-MYC translation. Further experiments are underway to test the potential of candidate miRNA and EIF4E3 as vincristine sensitizer in vivo. In conclusion, this study may identify novel factors that have potential not only to decrease the current therapeutic dose of vincristine and therefore eliminate its side effects, but also have potential to multiply the efficacy of lower doses in order to overcome hard to treat high-risk tumors. Citation Format: Nourhan Abdelfattah, Panneerdoss Subbarayalu, Benjamin Onyeagucha, Subapriya Rajamanickam, Hung-I Harry Chen, Manjeet Rao. MicroRNAs as novel therapeutic adjuvants to treat high-risk medulloblastoma.. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B01.

β2-adrenergic receptor signaling promotes pancreatic ductal adenocarcinoma (PDAC) progression through facilitating PCBP2-dependent c-myc expression

The β2-adrenergic receptor (β2-AR) plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) progression. In this report, we identified poly(rC)-binding protein 2 (PCBP2) as a novel binding partner for β2-AR using immunoprecipitation-mass spectrometry (IP-MS) approach. The association between β2-AR and PCBP2 was verified using reciprocal immunoprecipitation. Importantly, we found significant interaction and co-localization of the two proteins in the presence of β2-AR agonist in Panc-1 and Bxpc3 PDAC cells. β2-AR-induced recruitment of PCBP2 led to augmented protein level of c-myc in PDAC cells, likely as a result of enhanced internal ribosome entry segment (IRES)-mediated translation of c-myc. The activation of β2-AR accelerated cell proliferation and colony formation, while knockdown of PCBP2 or c-myc restrained the effect. Furthermore, overexpression of PCBP2 was observed in human PDAC cell lines and tissue specimens compared to the normal pancreatic ductal epithelial cells and the non-cancerous tissues respectively. Overexpression of β2-AR and PCBP2 was associated with advanced tumor stage and significantly worsened prognosis in patients with PDAC. Our results elucidate a new molecular mechanism by which β2-AR signaling facilitates PDAC progression through triggering PCBP2-dependent c-myc expression.

Disruption of the mTOR-eIF4F Axis By Selectively Targeting PI3Kdelta and Proteasome Potently Inhibits Cap Dependent Translation of c-Myc in Aggressive Lymphomas

Background: c-Myc is one of the most frequently altered genes across a vast array of human cancers. Overexpression of c-Myc is observed in up to 30% of cases of diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive lymphoma. Although DLBCL can be cured in 60-70% patients, a substantial minority of patients with DLBCL still die from their lymphoma. There is emerging evidence that c-Myc expression is an adverse risk factor independent of the cell-of-origin classification. To date no drugs that directly target c-Myc have been approved for the treatment of any cancer. In fact, since c-Myc is involved in many normal cellular functions, direct c-Myc inhibitors may be associated with significant toxicity. The goal of our study is to develop novel strategies targeting c-Myc that will have an improved therapeutic index. The c-Myc protein has a very short half-life of less than 30 minutes, and its translation is highly dependent on the eukaryotic initiation factor 4F (eIF4F). eIF4F is activated by the mammalian target of rapamycin (mTOR), which is regulated not only by the PI3K-AKT pathway but also the proteasome pathway. These observations led us to hypothesize that if the proteasome and PI3K pathways cooperate in the activation of mTOR and its downstream target eIF4F, then combinations of proteasome and PI3K inhibitors should potently suppress eIF4F dependent translation of c-Myc and the growth of c-Myc dependent lymphoma.Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo. The Bliss additivism model was used to determine the expected inhibition of cell growth. The difference of the expected and observed levels of inhibition was used to calculate the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Mechanisms of synergy were determined through interrogation of PI3K/AKT/mTOR pathway and its downstream targets. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups.Results: We used a high-throughput platform to screen the cytotoxicity of two PI3Kdelta inhibitors (TGR-1202 & idelalisib/Cal-101), two proteasome inhibitors (bortezomib & carfilzomib), and four combination pairs using these drugs. We found that TGR-1202 and Cal-101 caused only minimal to mild inhibition of lymphoma cells, while bortezomib and carfilzomib caused potent inhibition as single agents. The combination of TGR-1202 and carfilzomib was consistently the most synergistic doublet, while the combination of Cal-101 and bortezomib the least synergistic in numerous lymphoma cell lines studied to date (Figure 1). TGR-1202 and carfilzomib were also highly synergistic in primary lymphoma cells, while Cal-101 and bortezomib were not. Importantly, normal lymphocytes were resistant to the combination of TGR-1202 and carfilzomib. At the molecular level, only the combination of TGR-1202 and carfilzomib potently inhibited mTORC1 dependent phosphorylation of 4E-BP1, leading to marked reduction of c-Myc protein (Figures 2 & 3). In contrast, the combination of TGR-1202 and carfilzomib produced no reduction of the mRNA level of c-Myc (Figures 3). A luciferase reporter demonstrated that the synergistic combination TGR-1202 and carfilzomib specifically inhibited the translation downstream of the 5'UTR of c-Myc (Figure 3). GEP studies confirmed that the canonical c-Myc target genes were potently downregulated at the level of transcription by the combination of TGR-1202 and carfilzomib (Figure 3). These results demonstrate that TGR-1202 and carfilzomib in combination potently inhibited the translation of c-Myc and the c-Myc transcriptional program, which appears to be primarily through disruption of the PI3Kdelta and proteasome pathways that converge on mTOR. Ongoing experiments are focused on confirmation of these observations in vivo. Further, a phase I/II clinical trial evaluating this combination regimen in aggressive c-Myc driven lymphomas is being planned. [Display omitted] [Display omitted] [Display omitted] DisclosuresDeng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Lentzsch:Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Axiom: Honoraria. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria.

Identification of a Meta-Gene Network Associated with Metformin Sensitivity and Clinical Outcomes in Double Hit and Double Expressor Lymphomas

▪BackgroundThe "double hit" (DH) lymphomas that harbor a c-myc mutation and BCL2 translocation, or "double protein expressor" (DP) lymphomas that over-express c-myc and BCL2 proteins in the absence of a detectable mutation, have amongst the worst clinical outcomes as compared to patients with diffuse large B-cell lymphomas (DLBCL) that lack upregulation of the c-myc oncogene. Metformin can down-regulate translation of c-myc, making it an appropriate anti-cancer drug to explore in c-myc+ lymphomas. Furthermore, amethod to identify DH/DP patients most likely to benefit from metformin treatment has clinical relevance.MethodsWithin a publicly available gene expression array data set of R-CHOP treated DLBCL (n=232; GSE10846), a subset of DH/DP patients were defined as having above median expression of myc and BCL2 and below median expression of BCL6 as previously published by Horn et al. Survival analysis, significance analysis of microarrays (SAM) and gene set analysis (GSA) were performed characterizing the clinical, individual gene and biological ontology differences between DH/DP and non-DH/DP populations. Expression array data from a study testing metformin effects on THP-1 monocyte cells was reanalyzed using SAM and GSA as well. Changes in individual gene expression and overlapping ontological themes common to both GSA analyses of metformin effects on THP-1 cells and DH/DP characterization were identified. Genes with differential expression (DE) in both groups were evaluated topologically using a protein-protein interaction database to determine if any gene products had previously observed direct interactions. Network community detection identified tightly coupled signaling modules linking co-expression to mechanism. The resulting metformin-DH/DP network metagene was evaluated by k-means, clustering tumor samples into two groups over the metagene members in an independent data set of R-CHOP treated DLBCL patients (n=249; GSE32918) with differences in overall survival (OS) determined by log-rank.ResultsOf the 232 DLBCL patients treated with R-CHOP, 26 fit the criteria for DH/DP and had significantly lower OS (HR = 2.96; p < 0.001). In DH/DP tumors, 2780 genes had DE (2208 up-regulated; 572 down-regulated), enriched for biological processes related to transcription, metabolism and cytokine production and down-regulated for processes related to immune response, cell signaling, vascular development and proliferation (Fig. 1A). Analysis of metformin treated THP-1 cells relative to control identified 7123 genes with DE. Biological themes common to metformin treatment and DH/DP specific biology were identified including mitochondrial biogenesis, alternate splicing, and hormone secretion (Fig. 1A-B; highlighted in red). The intersection of genes with DE in metformin treated and DH/DP data sets identified 102 genes with direct interaction within a protein interaction network. Of the 19 communities detected by analyzing the resulting network topology, 3 showed significant correlation to survival in the GSE10846 data set (Fig. 2A, in red), forming a metformin-DH/DP metagene (Met-DH/DP-MG; n = 29 genes total). This metagene was validated by applying it to an independent cohort of R-CHOP treated DLBCL patients (n = 249), demonstrating 2 cluster groups (cluster 1, n=178; cluster 2, n=71; Fig. 2B) with differences in OS (HR = 1.61; p < 0.001; Fig. 2C).ConclusionWe have identified a metagene of interacting proteins associated with both metformin therapeutic effect and OS in DH/DP patients. This offers a potential method for selecting patients most likely to benefit from metformin therapy and identifies mechanistic avenues by which metformin treatment may specifically benefit DH/DP patients. As such, in vitro studies using DH cell lines and a phase I/II clinical trial exploring chemo-immunotherapy with metformin as an adjunct in DH/DP lymphomas are underway. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MTOR Dual Inhibitor Induced Cytotoxicity Depends on 4EBP1/c-Myc/Puma and NFkB/Egr-1/Bim Pathways in Human Lymphoid Malignancies

The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Src Inhibition Blocks c-Myc Translation and Glucose Metabolism to Prevent the Development of Breast Cancer

Preventing breast cancer will require the development of targeted strategies that can effectively block disease progression. Tamoxifen and aromatase inhibitors are effective in addressing estrogen receptor-positive (ER(+)) breast cancer development, but estrogen receptor-negative (ER(-)) breast cancer remains an unmet challenge due to gaps in pathobiologic understanding. In this study, we used reverse-phase protein array to identify activation of Src kinase as an early signaling alteration in premalignant breast lesions of women who did not respond to tamoxifen, a widely used ER antagonist for hormonal therapy of breast cancer. Src kinase blockade with the small-molecule inhibitor saracatinib prevented the disorganized three-dimensional growth of ER(-) mammary epithelial cells in vitro and delayed the development of premalignant lesions and tumors in vivo in mouse models developing HER2(+) and ER(-) mammary tumors, extending tumor-free and overall survival. Mechanistic investigations revealed that Src blockade reduced glucose metabolism as a result of an inhibition in ERK1/2-MNK1-eIF4E-mediated cap-dependent translation of c-Myc and transcription of the glucose transporter GLUT1, thereby limiting energy available for cell growth. Taken together, our results provide a sound rationale to target Src pathways in premalignant breast lesions to limit the development of breast cancers.

MiR-17-92 fine-tunes MYC expression and function to ensure optimal B cell lymphoma growth

The synergism between c-MYC and miR-17-19b, a truncated version of the miR-17-92 cluster, is well-documented during tumor initiation. However, little is known about miR-17-19b function in established cancers. Here we investigate the role of miR-17-19b in c-MYC-driven lymphomas by integrating SILAC-based quantitative proteomics, transcriptomics and 3′ untranslated region (UTR) analysis upon miR-17-19b overexpression. We identify over one hundred miR-17-19b targets, of which 40% are co-regulated by c-MYC. Downregulation of a new miR-17/20 target, checkpoint kinase 2 (Chek2), increases the recruitment of HuR to c-MYC transcripts, resulting in the inhibition of c-MYC translation and thus interfering with in vivo tumor growth. Hence, in established lymphomas, miR-17-19b fine-tunes c-MYC activity through a tight control of its function and expression, ultimately ensuring cancer cell homeostasis. Our data highlight the plasticity of miRNA function, reflecting changes in the mRNA landscape and 3′ UTR shortening at different stages of tumorigenesis.

Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress.

Protein translation is inhibited by the unfolded protein response (UPR)-induced eIF-2α phosphorylation to protect against endoplasmic reticulum (ER) stress. In addition, we found additional inhibition of protein translation owing to diminished mTORC1 (mammalian target of rapamycin complex1) activity in ER-stressed multiple myeloma (MM) cells. However, c-myc protein levels and myc translation was maintained. To ascertain how c-myc was maintained, we studied myc IRES (internal ribosome entry site) function, which does not require mTORC1 activity. Myc IRES activity was upregulated in MM cells during ER stress induced by thapsigargin, tunicamycin or the myeloma therapeutic bortezomib. IRES activity was dependent on upstream MAPK (mitogen-activated protein kinase) and MNK1 (MAPK-interacting serine/threonine kinase 1) signaling. A screen identified hnRNP A1 (A1) and RPS25 as IRES-binding trans-acting factors required for ER stress-activated activity. A1 associated with RPS25 during ER stress and this was prevented by an MNK inhibitor. In a proof of principle, we identified a compound that prevented binding of A1 to the myc IRES and specifically inhibited myc IRES activity in MM cells. This compound, when used alone, was not cytotoxic nor did it inhibit myc translation or protein expression. However, when combined with ER stress inducers, especially bortezomib, a remarkable synergistic cytotoxicity ensued with associated inhibition of myc translation and expression. These results underscore the potential for targeting A1-mediated myc IRES activity in MM cells during ER stress.

Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells.

RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1.

MicroRNA 363 mediated positive regulation of c-myc translation affect prostate cancer development and progress.

Prostate cancer (CaP) is the sixth most significant cancer killer of men in China. In this study, the potential role of micro-363 (miR-363) in CaP development and progression was investigated. Pri-miR-363 or anti-miR-363 was transfected into the CaP cells line PC-3 cells. Cell proliferation, transformation property, and epithelial-to-mesenchymal transition (EMT) were evaluated by MTT, clonogenic assay, colony formation in soft agar and western blotting, respectively. The expression and involvement of c-myc, adownstream target of miR-363 were also determined. The results showed that endogenous expression of miR-363 was significantly increased in CaP cells compared with normal prostate cells. High expression of miR-363 in PC-3 cells through transfection induces cell proliferation and positively regulates cell transformation property as well as promotes EMT of PC-3 cells. Through knockdown of c-myc, the results also showed that c-myc was involved in the regulation of biological function of PC-3 cells by miR-363. Taken together, this study adds support to the potential role of miR-363 in the diagnosis and treatment of CaP. Prostate cancer, transformation property, proliferation, micro-363, epithelial-to-mesenchymal transition.