MYC mRNA Levels Research Articles

Abstract 1343: DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization

Abstract Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with epithelial-to-mesenchymal transition (EMT) and enhanced tumor invasiveness and metastasis. However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. In fact, recent studies have indicated that nuclear localization of EGFR in cancer cells is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src-dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells, which possess EMT-like features and in which DUOX1 is normally silenced, results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression in A549 cells also suppressed EGF-stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Since both EGFR and Src are subject to redox regulation by cysteine oxidation, we are currently aiming to elucidate the molecular mechanisms by which these mechanism are affected by altered DUOX1 status. Citation Format: Andrew C. Little, Karamathullah Danyal, David Heppner, Milena Hristova, Albert van der Vliet. DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2017-1343

Abstract 2542: MSI2 binds LncRNAs and promotes self-renewal and oncogenesis through MYC expression

Abstract Backgrounds and Aims: RNA-binding protein MSI2 is elevated in several cancers and is linked to poor prognosis. Here, we sought to elucidate the role of MSI2 in regulating the expression of proto-oncogenes or tumor suppressor genes in hepatocellular carcinoma (HCC). Methods: We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs) and subsequent validation by qPCR analysis. Results: Among the MSI2-bound RNAs, MYC mRNA and long noncoding RNA (LncRNA) miR22 host gene (MIR22HG) and MARAT1 were identified by RIP-qPCR analysis. MSI2-MARAT1 binding dereppresses polycomb reppressor to recruit E2F1 in MYC promoter regions. It is known that the 5’-untranslated region of MYC contains an internal ribosome entry sequence (IRES) which allows IRES-dependent translation initiation along with canonical cap-dependent mechanisms. Our data show that MSI2 acts as an IRES trans-acting factors for MYC mRNA translation. MSI2 does not have any effect on the steady state level of MYC mRNA but significantly increases MYC protein levels, indicating that MSI2 regulates MYC at the post-transcriptional level. Further, we observed that MSI2 reduced the level of mature miR22 derived from MIR22HG processing. We showed that miR22 suppresses the expression of MYC thorough a 3’-UTR dependent process. Overexpression of MSI2 promotes TIC self-renewal and tumor initiation property in xenograft tumor mouse models, whereas silencing MSI2 reduces this occurrence. Conclusion: we demonstrate that MSI2 promotes liver tumorigenesis by maintenance of MYC expression through dereppression of polycomb repressor and an IRES-dependent translation mechanism and possibly by inhibition of miR22 processing. Thus, MSI2 may be a useful prognostic factor for HCC and MSI2 targeted therapy and thus may be beneficial in the treatment of HCC patients. Citation Format: Hifzur Siddique, Padmani Narayan, Vasu Punj, Douglas E. Feldman, Keigo Machida. MSI2 binds LncRNAs and promotes self-renewal and oncogenesis through MYC expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2017-2542

RNA Polymerase I Inhibition with CX-5461 as a Novel Therapeutic Strategy to Target MYC in Multiple Myeloma.

Dysregulation of MYC is frequently implicated in both early and late myeloma progression events, yet its therapeutic targeting has remained a challenge. Among key MYC downstream targets is ribosomal biogenesis, enabling increases in protein translational capacity necessary to support the growth and self-renewal programmes of malignant cells. We therefore explored the selective targeting of ribosomal biogenesis with the small molecule RNA polymerase (pol) I inhibitor CX-5461 in myeloma. CX-5461 induced significant growth inhibition in wild-type (WT) and mutant TP53 myeloma cell lines and primary samples, in association with increases in downstream markers of apoptosis. Moreover, Pol I inhibition overcame adhesion-mediated drug resistance and resistance to conventional and novel agents. To probe the TP53-independent mechanisms of CX-5461, gene expression profiling was performed on isogenic TP53 WT and knockout cell lines and revealed reduction of MYC downstream targets. Mechanistic studies confirmed that CX-5461 rapidly suppressed both MYC protein and MYC mRNA levels. The latter was associated with an increased binding of the RNA-induced silencing complex (RISC) subunits TARBP2 and AGO2, the ribosomal protein RPL5, and MYC mRNA, resulting in increased MYC transcript degradation. Collectively, these studies provide a rationale for the clinical translation of CX-5461 as a novel therapeutic approach to target MYC in myeloma.

Targeting of Wnt/β-Catenin by Anthelmintic Drug Pyrvinium Enhances Sensitivity of Ovarian Cancer Cells to Chemotherapy.

BackgroundAberrant activation of Wnt/β-catenin has been shown to promote ovarian cancer proliferation and chemoresistance. Pyrvinium, an FDA-approved anthelmintic drug, has been identified as a potent Wnt inhibitor. Pyrvinium may sensitize ovarian cancer cells to chemotherapy.Material/MethodsThe effect of pyrvinium alone and its combination with paclitaxel in ovarian cancer was investigated using an in vitro culture system and in vivo xenograft models. The mechanisms of its action were also analyzed, focusing on the Wnt/β-catenin pathway.ResultsPyrvinium inhibited growth and induced apoptosis of paclitaxel- and cisplatin-resistant epithelial ovarian cancer cell lines A2278/PTX and SK-OV-3. Its combination with paclitaxel was synergistic in targeting ovarian cancer cells in vitro. In 3 independent ovarian xenograft mouse models, pyrvinium alone inhibited tumor growth. More importantly, we observed significant inhibition of tumor growth throughout the treatment when using pyrvinium and paclitaxel combined. Mechanistically, pyrvinium increased the Wnt-negative regulator axin and decreased the β-catenin levels in ovarian cancer cells. In addition, pyrvinium suppressed Wnt/β-catenin-mediated transcription, as shown by the decreased mRNA levels of MYC, cyclin D, and BCL-9. In contrast, the inhibitory effects of pyrvinium were reversed by β-catenin stabilization or overexpression, demonstrating that pyrvinium acted on ovarian cancer cells via targeting the Wnt/β-catenin signaling pathway.ConclusionsWe demonstrated that the anthelmintic drug pyrvinium targets ovarian cancer cells through suppressing Wnt/β-catenin signaling. Our work highlights the therapeutic value of inhibiting Wnt/β-catenin in ovarian cancer.

Concurrent Targeting of BCL2 and MYC Transcription Leads to Chemo-Sensitization of Dual-Expressing Diffuse Large B-Cell Lymphoma In Vivo

To date, there are no effective strategies to treat patients with a particularly lethal form of diffuse large B-cell lymphoma (DLBCL) that over-express both B-cell lymphoma gene-2 (BCL2) and cellular-myelocytomatosis (MYC) oncogenes. Here, we provide a novel approach for simultaneously targeting BCL2 and MYC directly through secondary DNA structures. These structures act as molecular switches, turning gene expression on or off, and serve as scaffolds for protein and small molecule interaction. Previously identified compounds IMC-76 and GQC-05, recognize the BCL2 or MYC promoter region DNA structures, respectively, and independently lower mRNA and protein expression of each oncogene.For utility in sensitizing aggressive lymphoid tumors that concurrently dual express BCL2 and MYC, we examined the efficacy ofIMC-76 and GQC-05 in the presence and absence of a standard chemotherapy, cyclophosphamide (CPA)to inhibit tumor growth in a severe combinedimmunodeficient U2932 DLBCLxenograft mouse model.Unlike IMC-76, effects of GQC-05 in a xenograftmouse model are unknown; therefore, our initial in vivo studies involved GQC-05 alone and with IMC-76 in combination with CPA to determine the concentration for MYCdown-regulation and a maximum tolerated dose (MTD). At 2.5 and 5 mg/kg GQC-05 with 50 mg/kg CPA, we observed no effect on mean mouse weight and while the 5 mg/kg GQC-05-CPA combination group exhibited some mouse death, LD50 was not reached. Tumors from mice treated with GQC-05 and CPA were compared to diluent or CPA alone treated mice for MYC mRNA level and found to have significantly less MYC expression. As expected, there was no effect on BCL2 mRNA levels. Next, we carried over the 5 mg/kg GQC-05 to a subsequent in vivo experiment where we also tested a 10 mg/kg GQC-05 dose and included IMC-76 at 10 mg/kg, a previously established efficacious dose, to determine if we would reach an MTD, but still retain the repression of MYC. While the mice were able to tolerate the 5 mg/kg GQC-05 dose when IMC-76 and/or CPA was added to the treatment regimen, the LD50 was reached and surpassed in mice treated with the 10 mg/kg of GQC-05 combinations. However, both concentrations of GQC-05 when co-treated with IMC-76 and CPA resulted in a notable decrease of tumor MYC expression relative to untreated and CPA only treated mice as well as a detectable inhibition of BCL2.We then conducted a follow-up study to evaluate effects on tumor burden using concentrations of GQC-05 below the LD50, 5 mg/kg and 7.5 mg/kg, and also reduced the dose of CPA to 30 mg/kg. The co-treatment of mice with IMC-76 (10 mg/kg) and GQC-05 (7.5 mg/kg) in combination with CPA delayed tumor growth and decreased tumor size by 50%, 46%, and 37% at 9, 12, and 15 days post-first-day drug administration compared to the diluent and 50%, 67%, and 44% smaller tumors relative to CPA only treated mice (Figure 1). Overall, there was a prominent decrease in tumor burden in these mice as determined by area under the curveand the tumors displayed a lower expression of BCL2 and MYC at both the mRNA and protein levels. Interestingly, tumors of mice that received IMC-76 and GQC-05 also showed knock-down of BCL2 and MYC, but no effect on tumor burden unless CPA was present indicating these transcriptional inhibitors act as chemo-sensitizers. None of the various drug regimens resulted in a significant loss of mouse weight and while survival for GQC-05 7.5 mg/kg treated mice was reduced, LD50 was not reached.These are the first studies to demonstrate two differentpromoterDNA secondary structures can be targeted at the same time for direct transcriptional inhibition that leads tochemo-sensitization and slowed tumor growth in mice.Our findings demonstrate the potential utility of a dual-targeted, precision medicine-based strategy to improve the response of DLBCL patients to current chemotherapy and overcome resistant disease. [Display omitted] DisclosuresGokhale:Tetragene: Equity Ownership. Hurley:Tetragene: Consultancy, Equity Ownership. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

304 - DUOX1 Silencing in Lung Cancer is Associated with Enhanced Nuclear EGFR Localization

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H 2 O 2 producing enzyme located in the airway epithelium with key roles in epithelial homeostasis. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with features of the epithelial-to-mesenchymal transition (EMT). However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently misregulated in cancer. In fact, recent studies have indicated that nuclear EGFR in cancer is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src-dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells (low DUOX1), results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression also suppressed EGF-stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Funding provided by NIH RO1 HL085646 (AvdV), F32 HL129706 (DEH), T32 HL076122 (ACL), and LCCRO Juckett Award.

Abstract 3022: BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML)

Abstract PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc, plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report the preclinical activity of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor (AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02) currently being investigated in Phase I clinical trials in advanced cancer (NCT01938638) and acute leukemia (NCT02345382) in various in vitro, ex vivo and in vivo models of AML. BAY 1143572 inhibited the proliferation of 7 MLL-rearrangements positive and negative AML cell lines with a median IC50 of 385 nM (range 230-1100 nM) and induced apoptosis. Furthermore, BAY 1143572 showed potent in vitro activity in 8 out of 10 non-MLL-rearranged patient derived AML samples incl. NPM1 mutant and Flt3-ITD positive samples derived from intermediate and high risk patients. Moreover, we elucidated the dynamic changes of the cellular proteome/phosphoproteome upon pharmacological and genetic PTEFb inhibition and identified PTEFb interaction partners in various AML in vitro models. These analyses uncover the oncogenic PTEFb-dependent signaling networks and substantiate the molecular rationale for the use of PTEFb inhibitors in this indication. When applied in vivo, BAY 1143572 exhibited single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats upon once daily oral administration. Of note, partial or even complete remissions could be achieved in several models. Furthermore, intermittent dosing schedules with up to 4 days treatment pauses were feasible in terms of efficacy and tolerability. Using MOLM-13 xenografts in mice and rats to address the in vivo MoA of BAY 1143572, a transient inhibition of RNA polymerase II phosphorylation, MYC mRNA and protein levels, MCL-1 mRNA and protein levels, and an induction of apoptosis was documented. In conclusion, our data provide the rationale for the initiation of clinical development of BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential for the treatment of AML patients. A phase I clinical trial to determine the safety, tolerability and recommended Phase 2 dose in this indication is ongoing (NCT02345382). Citation Format: Arne Scholz, Thomas Oellerich, Akhtar Hussain, Sarah Lindner, Ulrich Luecking, Annette O. Walter, Peter Ellinghaus, Ray Valencia, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Stuart Ince, Hubert Serve, Karl Ziegelbauer. BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3022.

P62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA

Aberrant p62 overexpression has been implicated in breast cancer development. Here, we found that p62 expression was elevated in breast cancer stem cells (BCSCs), including CD44+CD24− fractions, mammospheres, ALDH1+ populations and side population cells. Indeed, short-hairpin RNA (shRNA)-mediated knockdown of p62 impaired breast cancer cells from self-renewing under anchorage-independent conditions, whereas ectopic overexpression of p62 enhanced the self-renewal ability of breast cancer cells in vitro. Genetic depletion of p62 robustly inhibited tumor-initiating frequencies, as well as growth rates of BCSC-derived tumor xenografts in immunodeficient mice. Consistently, immunohistochemical analysis of clinical breast tumor tissues showed that high p62 expression levels were linked to poorer clinical outcome. Further gene expression profiling analysis revealed that p62 was positively correlated with MYC expression level, which mediated the function of p62 in promoting breast cancer stem-like properties. MYC mRNA level was reduced upon p62 deletion by siRNA and increased with p62 overexpression in breast cancer cells, suggesting that p62 positively regulated MYC mRNA. Interestingly, p62 did not transactivate MYC promoter. Instead, p62 delayed the degradation of MYC mRNA by repressing the expression of let-7a and let-7b, thus promoting MYC mRNA stabilization at the post-transcriptional level. Consistently, let-7a and let-7b mimics attenuated p62-mediated MYC mRNA stabilization. Together, these findings unveiled a previously unappreciated role of p62 in the regulation of BCSCs, assigning p62 as a promising therapeutic target for breast cancer treatments.

262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.

PD32-01 PRE-CLINICAL RATIONALE FOR COMBINATION PI3K AND BRD4 INHIBITION IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-01 PRE-CLINICAL RATIONALE FOR COMBINATION PI3K AND BRD4 INHIBITION IN ADVANCED PROSTATE CANCER Paul Toren, Jared Allman, Soojin Kim, and Amina Zoubeidi Paul TorenPaul Toren More articles by this author , Jared AllmanJared Allman More articles by this author , Soojin KimSoojin Kim More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.677AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. This study details our pre-clinical evaluation of AZD8186, a novel β- and δ- selective PI3K small molecule inhibitor in prostate cancer models. We and others have recently noted the role of the oncogene myc as a mechanism of resistance to monotherapy treatment with PI3K/Akt inhibitors. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. METHODS Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCR were used to measure AR and myc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. RESULTS LNCaP cells demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo, castrate resistant LNCaP xenografts demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186. On-target decreases in pAkt was observed in xenograft tumour samples by western blot analysis. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in prostate cancer cell lines and xenograft tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the elevation of myc transcript levels induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR transcript levels. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. CONCLUSIONS Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs and may result in treatment resistance. We demonstrate BRD4 inhibition using JQ1 as a rationale combination strategy with PI3K inhibition. This strategy co-targets both myc and AR feedback pathways and warrants further investigation in prostate cancers with an activated PI3K/Akt pathway. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e761 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Paul Toren More articles by this author Jared Allman More articles by this author Soojin Kim More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD32-03 A PHASE 2 OPEN-LABEL TRIAL OF GTX-758 IN MEN WITH CASTRATION-RESISTANT PROSTATE CANCER, FINAL ANALYSIS OF THE PRIMARY ENDPOINT

INTRODUCTION AND OBJECTIVES: The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. This study details our pre-clinical evaluation of AZD8186, a novel band dselective PI3K small molecule inhibitor in prostate cancer models. We and others have recently noted the role of the oncogene myc as a mechanism of resistance to monotherapy treatment with PI3K/Akt inhibitors. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. METHODS: Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCRwere used tomeasure ARandmyc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. RESULTS: LNCaP cells demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo, castrate resistant LNCaP xenografts demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186. On-target decreases in pAkt was observed in xenograft tumour samples by western blot analysis. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in prostate cancer cell lines and xenograft tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the elevation of myc transcript levels induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR transcript levels. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. CONCLUSIONS: Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs and may result in treatment resistance. We demonstrate BRD4 inhibition using JQ1 as a rationale combination strategy with PI3K inhibition. This strategy co-targets both myc and AR feedback pathways and warrants further investigation in prostate cancers with an activated PI3K/Akt pathway.

Inverse Relationship between Progesterone Receptor and Myc in Endometrial Cancer.

Endometrial cancer, the most common gynecologic malignancy, is a hormonally-regulated disease. Response to progestin therapy positively correlates with hormone receptor expression, in particular progesterone receptor (PR). However, many advanced tumors lose PR expression. We recently reported that the efficacy of progestin therapy can be significantly enhanced by combining progestin with epigenetic modulators, which we term “molecularly enhanced progestin therapy.” What remained unclear was the mechanism of action and if estrogen receptor α (ERα), the principle inducer of PR, is necessary to restore functional expression of PR via molecularly enhanced progestin therapy. Therefore, we modeled advanced endometrial tumors that have lost both ERα and PR expression by generating ERα-null endometrial cancer cell lines. CRISPR-Cas9 technology was used to delete ERα at the genomic level. Our data demonstrate that treatment with a histone deacetylase inhibitor (HDACi) was sufficient to restore functional PR expression, even in cells devoid of ERα. Our studies also revealed that HDACi treatment results in marked downregulation of the oncogene Myc. We established that PR is a negative transcriptional regulator of Myc in endometrial cancer in the presence or absence of ERα, which is in contrast to studies in breast cancer cells. First, estrogen stimulation augmented PR expression and decreased Myc in endometrial cancer cell lines. Second, progesterone increased PR activity yet blunted Myc mRNA and protein expression. Finally, overexpression of PR by adenoviral transduction in ERα-null endometrial cancer cells significantly decreased expression of Myc and Myc-regulated genes. Analysis of the Cancer Genome Atlas (TCGA) database of endometrial tumors identified an inverse correlation between PR and Myc mRNA levels, with a corresponding inverse correlation between PR and Myc downstream transcriptional targets SRD5A1, CDK2 and CCNB1. Together, these data reveal a previously unanticipated inverse relationship between the tumor suppressor PR and the oncogene Myc in endometrial cancer.

Abstract A39: A role for src kinase in estrogen receptor-positive breast cancer

Abstract Estrogen (E2) stimulation promotes proliferation in estrogen receptor-positive (ER+) breast cancer, which accounts for 60-75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and “non-genomic” signaling cascades, which do not involve the transcriptional activity of the ER. This “non-genomic” pathway likely involves Src family kinases (SFKs) and has been implicated in cell cycle progression in fibroblasts engineered to express the ER (Castoria 1999). While not typically amplified or mutationally activated in ER+ breast cancer, Src is hyperactive in most breast cancers. To test whether SFK activity is required for G1/S cell cycle progression in breast cancer cells, we pretreated quiescent ER+ MCF7 or ZR75-1 cells with a selective SFK inhibitor (SU11333) prior to stimulation with E2. Inhibition of SFK activity blocked progression from G1 to S phase. Previous data from our laboratory has demonstrated that G1/S progression in response to peptide growth factor stimulation of quiescent fibroblasts is dependent upon SFK-mediated stabilization of myc mRNA (Bromann 2005). We therefore tested whether SFK activity also regulates myc expression in breast cancer cells. Our data suggest that SFK activity is required for accumulation of myc mRNA in quiescent MCF7s after E2 stimulation, and that E2 stimulated stabilization of myc mRNA is dependent on SFK activity. We are currently investigating the mechanism involved in stabilizing myc mRNA levels.

Pre-clinical rationale for combination PI3K and BRD4 inhibition in advanced prostate cancer.

234 Background: The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. Here we detail our pre-clinical evaluation of AZD8186, a novel β and δ selective PI3K small molecule inhibitor. Further, we investigate how increased transcription of the myc oncogene may represent a mechanism of resistance to monotherapy PI3K/Akt inhibition. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. Methods: Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCR were used to measure AR and myc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. Results: In vitro results demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo results demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186 with on-target decreases in pAkt was observed in xenograft tumour samples. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in cells and tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the increase of myc induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. Conclusions: Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs. We demonstrate BRD4 inhibition using JQ1 which co-targets both myc and AR feedback pathways as a rationale combination strategy with PI3K inhibition. This strategy warrants further investigation in prostate cancers with an activated PI3K/Akt pathway.

Ethylenediaminetetraacetic Acid, Sodium Citrate, Heparin and Citrate Phosphate Dextrose-Adenine Anticoagulants Differentially Affect Cytokine mRNA Expression in Blood Leukocytes.

The aim of this study is to determine whether and how the anticoagulant agents EDTA, sodium citrate, and heparin present in vacutainers and CPDA located in transfusion bags modify peripheral blood cell behavior. We compared the effect of four anticoagulants within an expression study quantifying mRNA by realtime PCR in 60 blood samples. We observed a significant increase of TNFα and CCL2 mRNA expression in leukocytes placed in EDTA anticoagulant compared to leukocytes collected with citrate or heparin (p ≤ 0.5). Compared to blood cells influenced by one of the three anticoagulant agents present in vacuum tubes, cells from transfusion bags affected by CPDA displayed significantly lower levels of CCL2 and MYC mRNA, whereas levels of IL-10 and TNFα mRNA were much higher (p ≤ 0.5). The anticoagulant EDTA significantly increases proinflammatory cytokine mRNA in freshly isolated PBMC. Anticoagulant CPDA significantly alters levels of some of the tested mRNAs in comparison to blood cells drawn into one of the three anticoagulant agents present in vacutainers.

Identification of the Epigenetic Reader BRD4 As a Novel Therapeutic Target in JAK2 V617F+ MPN Cells

Myeloproliferative neoplasms (MPN) are characterized by clonal expansion and accumulation of erythrocytes, platelets, and myeloid cells in the bone marrow (BM) and other organs. Classical MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation is frequently detected in neoplastic cells in patients with MPN. Although MPN are chronic and indolent diseases in most patients, fatal progression may occur. So far, the only curative approach for these patients is hematopoietic stem cell transplantation. Therefore, current research is evaluating new therapeutic targets and the effects of various targeted drugs. The epigenetic reader bromodomain-containing protein 4 (BRD4) has recently been identified as a promising target in acute myeloid leukemia. In the present study, we investigated the potential value of BRD4 as a molecular target in MPN. We employed two JAK2 V617F+ cell lines, SET-2 and HEL, as well as BM samples obtained from 18 MPN patients (ET: n=7; PV: n=7; PMF: n=4). Three BRD4 inhibitors were applied: JQ1, BI2536, and BI6727. As assessed by qPCR, primary MPN cells as well as SET-2 cells and HEL cells were found to express BRD4 mRNA. In 3 H-thymidine uptake experiments, all three BRD4 blockers were found to suppress the proliferation of the two MPN cell lines and of primary MPN cells in 8/8 patients tested. The effects of these drugs were dose-dependent, with the following IC50 values obtained in SET-2 cells: JQ1, 50-100 nM; BI2536, 20-40 nM; BI6727, 50-75 nM; and in HEL cells: JQ1, 100-500 nM; BI2536, 20-40 nM; BI6727, 30-50 nM. In primary MPN cells, all three agents tested produced IC50 values between 500 and 1000 nM. In normal BM cells, JQ1 did not produce a reasonable IC50 value (>5000 nM). In one patient sample (PMF), we analyzed the effect of JQ1 on the percentage of putative (neoplastic) stem cells (CD34+/CD38-). In this experiment, exposure to JQ1 was followed by a decrease in the percentage of CD34+/CD38- cells compared to control medium (control: 0.16% vs JQ1: 0.045%). To confirm the role of BRD4 as a potential target in MPN cells, we performed target-knockdown experiments in SET-2 cells and HEL cells using two different BRD4 shRNAs (#602 and #1817) and a random shRNA as control. In these experiments, the shRNA-induced knockdown of BRD4 was found to block proliferation in SET2 cells and HEL cells when compared to untransfected cells or random shRNA-transduced cells. In a next step, we examined the mechanism of drug-induced growth inhibition. In cell cycle experiments, BI2536 and BI6727 were found to induce a G2/M phase arrest in both cell lines. By contrast, JQ1 induced a G1 arrest in HEL cells, but did not show a significant effect on cell cycle progression in SET-2 cells. We also asked whether BRD4 inhibition is associated with induction of apoptosis in MPN cells. All three BRD4 blockers induced apoptosis in SET-2 cells and HEL cells at relatively high concentrations after 48 hours, with ED50 values of >5 µM for JQ1 and 0.5-5.0 µM for BI2536 and BI6727. Finally, we asked whether exposure to BRD4 inhibitors is associated with modulation of BRD4 mRNA or MYC mRNA expression. As assessed by qPCR, JQ1, BI2536, and BI6727 were found to downregulate BRD4 mRNA levels as well as MYC mRNA levels in SET-2 cells and HEL cells. In conclusion, our data show that BRD4 is expressed in JAK2 V617F+ MPN cells and that BRD4 inhibition is associated with decreased proliferation and survival of neoplastic cells. The clinical value of BRD4 as a novel target in MPN cells remains to be determined. DisclosuresZuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Staber:Genactis: Research Funding; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Abstract PR12: Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides

Abstract Despite its well-characterized properties as a proto-oncogene in numerous cancers, direct potent and selective inhibition of MYC or its protein product c-Myc with cell-permeable synthetic agents remains a significant challenge. Systemic inhibition of c-Myc activity in mice by expression of dominant negative MYC transgenes has been shown to dramatically decrease tumor burden with apparently tolerable side effects (Soucek et al, Nature 2008). In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC at the transcriptional, translational, and post-translational level. Here we describe the application of synthetic antisense oligonucleotides designed to target specific sequences of the MYC mRNA (MYCASOs). We have incorporated several chemical features into MYCASOs, including the addition of locked nucleic acid (LNA) bases at the 5' and 3' ends, phosphorothioate linkers, and internal DNA bases in order to increase mRNA target affinity and cleavage, cellular permeability, stability, and systemic distribution in vivo. Treatment of MYC¬-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and c-Myc protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 5' rapid amplification of cDNA ends (RACE) assay. MYCASO-treatment of cancer cell lines leads to significant inhibition of cellular proliferation and induces apoptosis in c-Myc-dependent myeloma and leukemia cell lines while decreasing c-Myc-mediated gene expression. In vivo administration of MYCASOs simply dissolved in saline was performed in mice by tail vein injection. MYCASOs are well tolerated up to 25 mg per kg on a twice-weekly dosing regimen. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment leads to detectable cleavage of the MYC transcript, decreases c-Myc levels within tumors, and significantly decreases tumor burden. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of c-Myc activity, and promising therapeutic agents for MYC-driven tumors. Citation Format: Christopher J. Ott, Raj Bandaru, Taylor Gill, Junyan Tao, Xin Chen, Yixian Zhang, James E. Bradner. Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR12.

Abstract DDT02-02: BAY 1143572: A first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, inhibits MYC and shows convincing anti-tumor activity in multiple xenograft models by the induction of apoptosis

Abstract PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like MYC, a key oncogene in multiple tumors, plays a critical role in cancer cell growth and survival. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors which are currently evaluated in Phase I and II clinical trials, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report for the first time the preclinical profile and structure of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor currently being investigated in a Phase I clinical trial. BAY 1143572 had potent and highly selective PTEFb-kinase inhibitory activity in the low nanomolar range against PTEFb/CDK9 and an at least 50-fold selectivity against other CDKs in enzymatic assays. Furthermore, BAY 1143572 showed a favorable selectivity against a panel of non-CDK kinases in vitro. The potent enzymatic activity on PTEFb translated into broad antiproliferative activity against a panel of tumor cell lines with sub-micromolar IC-50 values. In line with the proposed mode of action, a concentration-dependent inhibition of the phosphorylation of the RNA polymerase II and downstream reduction of MYC mRNA and protein levels was observed in vitro. This inhibition was accompanied by an induction of apoptosis in cellular assays. BAY 1143572 also showed single agent in vivo efficacy at tolerated doses in various xenograft tumor models in mice and rats upon once daily oral administration. Potent anti-tumor activity characterized with partial or even complete remissions could be documented in models showing different MYC gene alterations like amplifications and translocations. Treatment with BAY 1143572 resulted in a transient inhibition of intratumoral MYC mRNA and protein levels and an induction of apoptosis in these models. The inhibition of MYC mRNA was also observed in blood cells of BAY 1143572-treated rats indicating the potential clinical utility of MYC in blood cells as a pharmacodynamic marker in clinical development. The in vivo efficacy of BAY 1143572 was significantly enhanced in combination with several chemotherapeutics in different solid tumor models. These pharmacology data provided the rationale for the initiation of clinical development of BAY 1143572 in advanced cancer patients (NCT01938638). In conclusion, our data provide preclinical proof of concept for BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential. Further clinical evaluation of BAY 1143572 for the treatment of cancers dependent on the transcription of the key oncogene MYC and other short-lived survival proteins is warranted. Citation Format: Arne Scholz, Ulrich Luecking, Gerhard Siemeister, Philip Lienau, Ulf Boemer, Peter Ellinghaus, Annette O. Walter, Ray Valencia, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY 1143572: A first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, inhibits MYC and shows convincing anti-tumor activity in multiple xenograft models by the induction of apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02. doi:10.1158/1538-7445.AM2015-DDT02-02

Abstract 2612: BET-protein bromodomain antagonist-based combinations against ibrutinb-sensitive or resistant human Mantle Cell Lymphoma cells

Abstract Mantle Cell Lymphoma (MCL) cells demonstrate genetic alterations involving cyclin D1, CDK4, CDKN2A, MYC, BCL2, as well as in the Toll-like receptor, B-cell receptor (BCR) and NFkB signaling genes. Increased BCR signaling and transcriptional activity of NFkB is documented in MCL cells. Ibrutinib (IB) is an inhibitor of Bruton's tyrosine kinase (BTK) clinically effective against MCL, but long-term durable remissions and cure following IB treatment remains uncertain. We have previously reported that treatment with the BET protein bromodomain antagonist (BA) JQ1 disrupts the binding of the BET-protein BRD4 to the enhancers and promoters and inhibits the mRNA and protein levels of the oncogenes MYC, BCL-2 and CDK4/6 in MCL cells. BRD4 has also been shown to be essential for NFkB activity. Consistent with this, here, we determined that treatment with JQ1 (250 to 2000 nM) reduced the nuclear localization of RelA and attenuated the expression of the NFkB target genes, including TNFα, TNFAIP3 (A20), c-FLIP, cIAP2, XIAP and Bcl-xL in the cultured MCL Mino and MO2058 cells. Notably, JQ1 treatment also reduced the mRNA and protein levels of BTK, along with inhibition of the levels of p-BTK, p-PLCγ2 and p-AKT levels in the MCL cells. Concomitantly, JQ1 dose-dependently inhibited cell growth and exerted lethal activity against cultured (MO2058 and Mino, Z138 and JeKo-1) and patient-derived, primary MCL (pMCL) cells, while relatively sparing normal CD19+ B cells and CD34+ bone marrow progenitor cells. Importantly, co-treatment with JQ1 and IB also markedly inhibited the nuclear RelA levels and attenuated the levels of p-BTK and BTK and reduced NFkB target gene expressions. Co-treatment with JQ1 and IB also synergistically induced apoptosis of the cultured and pMCL cells (CI values < 1.0 by isobologram analysis). Following tail-vein infusion and engraftment of the Mino cells in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, IP) and IB (25 mg/kg/day IP) versus treatment with each agent alone significantly improved the median survival of the mice (p < 0.01). We isolated in vitro IB-resistant Mino/IR cells (> 20-fold resistant), following continuous exposure to IB. As compared to the parental control Mino, Mino/IR cells expressed markedly higher levels of AKT, CDK6, BCL2, Bcl-xL, and XIAP. JQ1 treatment (1.0 μM) was able to inhibit the growth and induce apoptosis of Mino/IR cells. Notably, co-treatment with JQ1 and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT199 (BCL2 antagonist), or carfilzomib (CZ) (proteasome inhibitor) synergistically induced apoptosis of not only Mino but also Mino/IR cells. Taken together, these findings identify BA-based rational combinations that need to be further evaluated for their in vivo efficacy against IB-sensitive and IB-resistant MCL. Citation Format: Baohua Sun, Bhavin Shah, Warren Fiskus, Jun Qi, Santhana G. T. Devaraj, Swaminathan P. Iyer, Sunil Sharma, James E. Bradner, Youli Zu, Kapil N. Bhalla. BET-protein bromodomain antagonist-based combinations against ibrutinb-sensitive or resistant human Mantle Cell Lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2015-2612

Anti-proliferative effects of γ-tocotrienol are associated with suppression of c-Myc expression in mammary tumour cells.

Aberrant c-Myc activity plays a central role in cancer transformation. γ-tocotrienol is a member of the vitamin E family that displays potent anti-cancer activity. Here, studies were conducted to determine the role of c-Myc in mediating anti-proliferative effects of γ-tocotrienol in mammary cancer cells. Treatment effects on mouse +SA and human MCF-7 mammary cancer cell proliferation were determined by MTT assay and Ki-67 staining. Protein expression was determined by western blot analysis. Immunofluorescence staining and qRT-PCR were used to characterize cellular c-Myc and MYC levels respectively. Anti-proliferative effects of γ-tocotrienol were associated with reduction in total c-Myc and phosphorylated-c-Myc-serine 62, and increase in phosphorylated-c-Myc-threonine 58 levels. γ-tocotrienol also reduced PI3K/Akt/mTOR and Ras/MEK/Erk mitogenic signalling, cyclin D1 and cyclin-dependent kinase 4 levels, and increased p27 levels. However, γ-tocotrienol had no effect on MYC mRNA levels. γ-tocotrienol also increased levels of FBW7 (E3 ligase that initiates ubiquitination of c-Myc), but had no effect on serine/threonine phosphatase PP2A or isomerase Pin 1 levels. Combined treatment with GSK3α/β inhibitor LiCl or proteasome inhibitor MG132 blocked γ-tocotrienol-induced reductions in c-Myc. These findings indicate that anti-proliferative effects of γ-tocotrienol are associated with reduction in c-Myc that results from increase in GSK-3α/β-dependent ubiquitination and degradation, rather than from reduction in c-Myc synthesis in +SA and MCF-7 mammary cancer cells.