Internal Ribosome Entry Sequence Research Articles

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients.A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Abstract 5335: circAde: a circRNA-based system for prolonged and more effective treatment of cancer

Abstract In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable. Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5335.

HnRNP-A1 binds to the IRES of MELOE-1 antigen to promote MELOE-1 translation in stressed melanoma cells.

The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)—coded for by a long noncoding RNA—whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1.

Analysis of Structure-Activity Relationships Based on the Hepatitis C Virus SLIIb Internal Ribosomal Entry Sequence RNA-Targeting GGHYRFK⋅Cu Complex.

New therapeutics for targeting the hepatitis C virus (HCV) have been released in recent years. Although they are less prone to resistance, they are still administered in cocktails as a combination of drugs targeting various aspects of the viral life cycle. Herein, we aim to contribute to an arsenal of new HCV therapeutics by targeting the HCV internal ribosomal entry sequence (IRES) RNA through the development of catalytic metallodrugs that function to degrade rather than inhibit the target molecule. Based on a previously characterized HCV IRES stem-loop IIb RNA-targeting metallopeptide Cu-GGHYrFK (1⋅Cu), an all-l analogue (3⋅Cu) and a series of additional complexes with single alanine substitutions in the targeting domain were prepared and screened to determine the influence each amino acid side chain on RNA localization and recognition, and catalytic reactivity toward the RNA. Additional substitutions of the tyrosine position in complex 3⋅Cu were also investigated. Good agreement between calculated and measured binding affinities provided support for in silico modeling of the SLIIb RNA binding site and correlations with RNA cleavage sites. Examination of the cleavage products from reaction of the Cu complexes with SLIIb provided mechanistic insights, with the first observation of the 5'-geminal diol and 5'-phosphopropenal as products through the use of a Cu⋅ATCUN catalytic motif. Together, the data yielded insights into structure-function relationships that will guide future optimization efforts.

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

Abstract 1614: Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response

Abstract The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines can result in serious adverse effects, often life-threatening. DNA transfer via electroporation (EP) is a safe and effective method of delivering cytokines to target tissues. Intratumoral (IT) electroporation of Interleukin 12 (IL-12), a potent immunomodulatory cytokine, is well tolerated with an acceptable safety profile and objective response rates of 34-40% and 33% in Phase II clinical trials in advanced melanoma and Merkel cell carcinoma, respectively. We sought to improve the systemic anti-tumor response of IT pIL-12-EP by improving IL-12p70 expression and electroporation conditions, which were evaluated in vitro and in vivo with a two-tumor syngeneic mouse model of melanoma. As functional IL-12 p70 is a heterodimer, we compared different expression constructs to achieve high levels of IL-12 protein expression. IL-12p70 protein expression from a plasmid that incorporated a picornavirus-derived co-translational cleavage site (P2A) was higher than constructs with an internal ribosomal entry sequence (IRES) or a fusion of the p35 and p40 subunits. In functional in vitro assays, pIL-12(P2A) was superior to pIL-12(IRES) and the fusion protein. Using the murine B16.F10 tumor model, we show that IT EP of pIL-12 (P2A) plasmid regresses the treated lesions in a dose-dependent manner compared to control treatments. Systemic effects of IT-expressed IL-12 was assessed by monitoring generation of an antigen-specific CD8 T cell response and regression of B16.F10 contralateral (untreated) tumors following primary tumor electroporation. IT-pIL12-EP treatment with the P2A-linked construct resulted in a significant increase in antigen-specific CD8 T cells, as well as enhanced contralateral tumor growth inhibition suggesting the induction of strong systemic anti-tumor immune response. * Contributed equally to this work Citation Format: David A. Canton, Christoph Burkart, Shawna A. Shirley, Anandaroop Mukhopadhyay, Richard J. Connolly, Arya Bahrami, Jean S. Campbell, Robert H. Pierce. Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response [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 1614. doi:10.1158/1538-7445.AM2017-1614

Genetic changes found in a distinct clade of Enterovirus D68 associated with paralysis during the 2014 outbreak.

Enterovirus D68 (EV-D68) caused a severe respiratory illness outbreak in the United States in 2014. Reports of acute flaccid myelitis (AFM)/paralysis (AFP) in several independent epidemiological clusters of children with detectable EV-D68 have raised concerns that genetic changes in EV-D68 could be causing increased disease severity and neurological symptoms. To explore the potential link between EV-D68 genetic variations and symptom changes, we performed a series of comparative genomic analyses of EV-D68 2014 outbreak isolate sequences using data and analytical tools in the Virus Pathogen Resource (ViPR; www.viprbrc.org). Our results suggest that (1) three distinct lineages of EV-D68 were co-circulating in 2013 and 2014; (2) isolates associated with AFM/AFP belong to a single phylogenetic subclade – B1; (3) the majority of isolates from the B1 subclade have 21 unique substitutions that distinguish them from other isolates, including amino acid substitutions in the VP1, VP2, and VP3 capsid proteins and the 3D RNA-dependent RNA polymerase, and nucleotide substitutions in the internal ribosome entry sequence (IRES); (4) at 12 of these positions, B1 isolates carry the same residues observed at equivalent positions in paralysis-causing enteroviruses, including poliovirus, EV-D70 and EV-A71. Based on these results, we hypothesize that unique B1 substitutions may be responsible for the apparent increased incidence of neuropathology associated with the 2014 outbreak.

HDMX Folds the Nascent p53 mRNA following Activation by the ATM Kinase

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

Replication Cycle and Molecular Biology of the West Nile Virus

West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s) used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3' and 5' sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5' RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated.

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

HnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.

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Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response

Little is known about either the basal or stimulated homeostatic mechanisms regulating nuclear tenure of Nf-e2-related factor 2 (Nrf2), a transcription factor that mediates expression of over 200 detoxification genes. Our data show that stress-induced nuclear Nrf2 accumulation is largely from de novo protein synthesis, rather than translocation from a pre-existing cytoplasmic pool. HepG2 cells were used to monitor nuclear Nrf2 24h following treatment with the dithiol micronutrient (R)-α-lipoic acid (LA; 50μM), or vehicle. LA caused a ≥2.5-fold increase in nuclear Nrf2 within 1h. However, pretreating cells with cycloheximide (50μg/ml) inhibited LA-induced Nrf2 nuclear accumulation by 94%. Providing cells with the mTOR inhibitor, rapamycin, decreased basal Nrf2 levels by 84% after 4h, but LA overcame this inhibition. LA-mediated de novo protein translation was confirmed using HepG2 cells transfected with a bicistronic construct containing an internal ribosome entry sequence (IRES) for Nrf2, with significant (P<0.05) increase in IRES use under LA treatment. These results suggest that a dithiol stimulus mediates Nrf2 nuclear tenure via cap-independent protein translation. Thus, translational control of Nrf2 synthesis, rather than reliance solely on pre-existing protein, may mediate the rapid burst of Nrf2 nuclear accumulation following stress stimuli.

Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35–45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp–2A–Cre and Flp–IRES–Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp–to–Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes.

Expression of Truncated Eukaryotic Initiation Factor 3e (eIF3e) Resulting from Integration of Mouse Mammary Tumor Virus (MMTV) Causes a Shift from Cap-dependent to Cap-independent Translation

Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes whereas mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.

Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid

IntroductionLocal synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably.MethodsTo follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5.ResultsMurine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth.ConclusionsOur modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.

Post-transcriptional and post-translational regulation of Bcl2

Bcl2 is an important pro-survival protein that has an essential function in normal immunity and whose constitutive expression leads to the development of lymphomas. Although transcriptional control of Bcl2 has been reported, increasing evidence suggests an important component of Bcl2 regulation is post-transcriptional. Phosphorylation of Bcl2 has been shown to enhance activity to allow response to extracellular growth-factor-mediated signals. Bcl2 mRNA contains regulatory elements in both its 5'- and 3'-UTRs (untranslated regions). An IRES (internal ribosome entry sequence) in the 5'-UTR permits continued translation in the presence of cellular stresses that reduce cap-dependent translation. The 3'-UTR of Bcl2 mRNA is 5.2kb in length and contains multiple predicted miRNA (microRNA) and RNA-BP (RNA-binding protein)-binding sites. miR-15a and miR-16-1 have been found to inhibit Bcl2 expression in B-cells, whereas the RNA-BP nucleolin has been shown to increase Bcl2 expression by binding to the 3'-UTR and enhancing mRNA stability. Both decreased expression of miR-15a and miR-16-1 and increased nucleolin have been shown to be associated with increased Bcl2 expression and resistance to apoptosis in the common human disease, chronic lymphocytic leukaemia. miRNA-based therapeutic approaches to treat cancer are emerging. Bcl2 is highly regulated by miRNAs and is therefore an excellent candidate for such approaches.

Transcriptional and Translational Regulation of the Cancer-Testis Antigen PRAME Mediated by Pathogen-Associated Molecular Patterns

AbstractAbstract 2475PRAME (preferentially expressed antigen in melanoma) is a germinal tissue-specific gene that is expressed at high levels in a number of haematological malignancies. In AML, PRAME is a positive prognostic indicator, being associated with a favourable response to chemotherapy and increased overall survival, even in the presence of unfavourable karyotypes. In contrast, over-expression of PRAME mRNA is associated with poor prognosis in CML, NHL, HD, CLL and myeloma, being associated with progressive disease and chemo-resistance. Despite proposals to implement PRAME as a tool for monitoring minimal residual disease and as an immunotherapeutic target, the physiological functions of PRAME in normal and tumour cells are unclear. A role in the regulation of retinoic acid signalling has been proposed, although interactions of PRAME with retinoic acid receptors are weak compared to other coregulator proteins. Sequence homology and structural predictions suggest that PRAME is a member of the family of leucine-rich repeat (LRR) proteins that function in cellular immunity, bearing most resemblance to the LRR domains of Toll-like receptors (in particular TLR3 and TLR4) and internalin proteins. LRR proteins can bind pathogen-associated molecular patterns (PAMPs) including proteins, nucleic acids and sugars, and consistent with this PRAME has been reported to interact with OPA proteins from Neisseriae gonorrhoeae. Here we show that expression of PRAME in HL60 leukaemic cells is strongly induced by lipopolysaccharide (LPS) or peptidoglycan in the presence of interferon (IFN)-gamma, whereas no induction was observed after exposure to other PAMPs such as zymosan A. This burst of PRAME expression peaked between 1 and 4 hrs post treatment, but was not mediated by the proximal promoter region. Moreover, we show that treatment of HL60 with LPS and IFN-gamma increases the association of PRAME transcripts with polysomes indicating PRAME is also translationally regulated, and we provide evidence that PRAME 5'UTR contains an internal ribosome entry sequence for CAP-independent translation. Finally, we show that the subcellular localisation of PRAME protein in HL60 and U937 cells is modulated by treatment with LPS and IFN-gamma. In summary, our studies indicate that the LRR protein PRAME is subject to transcriptional and translational regulation following cellular exposure to bacterial PAMPs and interferon, suggesting it may have a role in innate immunity. Disclosures:No relevant conflicts of interest to declare.

Genetic and Pharmacologic Targeting of STAT5/Gab2/PI3K/mTOR Signaling in a Mouse Myeloproliferative Disease Model.

Abstract 3902Poster Board III-838Signal transducer and activator of transcription-5 (STAT5) is a latent transcription factor that can be activated by phosphorylation by Janus kinases (JAKs) in the cytoplasm, leading to dimerization, DNA binding, and retention within the nucleus. Phosphorylated STAT5 as determined at the single cell level by flow cytometry is a biomarker associated with poor prognosis in certain types of myeloid malignancies including juvenile myelomonocytic leukemia and M4/M5 leukemias. However, targeting of phosphorylated STAT5 or its aberrant signaling might be difficult since direct inhibition of STAT5 transcriptional activity in its entirety may present significant side effects including immune suppression, hematopoietic stem cell dysfunction, or liver problems. Understanding aberrant STAT5 signaling in normal vs. leukemic cells may allow development of novel strategies for leukemia therapy. Our recent demonstrations of cytoplasmic localization of phosphorylated STAT5 in chronic myeloid leukemia and acute myeloid leukemia have lead us to explore to the efficacy of molecularly defining and targeting cooperating signaling pathways. Persistently active STAT5 (STAT5aS711F) has close encounters with Grb2-associated binding protein (Gab2) in the cytoplasm which is associated with increased activation of Akt. Enhanced sensitivity to inhibition of STAT5, SHP-2, and Gab2 has also been described in Bcr/Abl induced oncogenic activities. We have shown that STAT5aS711F expression induces myeloid expansion and promotes growth through a Gab2-dependent mechanism as determined in vitro using Gab2 decoy molecules. However, the potential efficacy of targeting Gab2 in vivo in a live animal disease model has not been explored. Here we have retrovirally transduced wild-type or Gab2-/- mouse bone marrow cells with a murine stem cell virus (MSCV)-based retroviral vector expressing STAT5aS711F upstream of an internal ribosomal entry sequence and the enhanced green fluorescent protein (GFP). Transplanted bone marrow cells caused the expected myeloproliferative disease (MPD) phenotypes that were quantified along with mouse survival. Mice reconstituted with transduced bone marrow cells lacking Gab2 had a significantly attenuated accumulation of myeloid Gr-1+/Mac-1+ cells in the peripheral blood (3.4-fold; P=0.014), liver, and spleen and showed improved survival (control 30 days vs. >90 days without Gab2). However, phosphorylation of Akt activated by STAT5aS711F also occurred in mice lacking Gab2, indicating that Gab2-independent activation of Akt also occurs. Like reported by others in Jak2 and Bcr/Abl mouse MPD models we observed cell extrinsic MPD suggesting that STAT5 activates a paracrine signaling pathway that does not depend on direct Gab2 interaction. This lead us to consider alternative approaches for targeting the PI3K/Akt pathway and to begin defining Akt target genes of importance in STAT5 provoked MPD. Rapamycin is an effective inhibitor of mTORC1 and has been previously shown to synergize with protein tyrosine kinase inhibitors. Therefore, we utilized rapamycin to test whether targeting PI3K/Akt/mTOR signaling would be sufficient to impair cytokine-independent growth provoked by STAT5aS711F. First, BaF3 cells expressing STAT5aS711F were tested for their responsiveness to rapamycin (0.01 – 1 nM). Treatment with rapamycin effectively stopped the cells from growing but was not cytotoxic. Due to the effects of rapamycin on hematopoietic engraftment, it was necessary to optimize transplant and disease burden for rapamycin treatment in vivo. Strikingly, daily treatment of transplanted mice (vehicle N=9; rapamycin N=10) with 4 mg/kg of rapamycin at the early stage of MPD provided a 3.3-fold reduction in Gr-1+/Mac-1+ cells (P=0.002) and prolonged survival of treated mice (>90 days with rapamycin compared with 60 days for control). This effect was cytostatic but did not prevent the subsequent recurrence of MPD once the treatment was stopped. The use of additional drugs and/or optimized transplant/treatment protocols will be required for future studies Overall, these data provide in vivo evidence for clinically significant cooperative signaling by STAT5 and AKT-mTOR pathway in leukemic hematopoiesis and support targeting of the Akt-mTOR axis as therapy for MPDs with Jak2 or Mpl mutations and phosphorylated STAT5. Disclosures:No relevant conflicts of interest to declare.

Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.