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Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and trans -amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in trans . To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes., Competing Interests: U.S., T.B., and A.Y. are inventors on patents and patent applications that cover parts of this article. U.S. is a management board member and employee and has securities from BioNTech SE, a company developing therapeutic RNA., (© 2024 The Authors.)

Alphaviruses such as the human pathogenic chikungunya virus (CHIKV) and Ross River virus (RRV) can cause explosive outbreaks raising public health concerns. However, no vaccine or specific antiviral treatment is yet available. We recently established a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). This novel system consists of a replicase-encoding mRNA and a trans-replicon (TR) RNA encoding the antigen. The TR-RNA is amplified by the replicase in situ. We were interested in determining whether multiple TR-RNAs can be amplified in parallel and if, thus, a multivalent vaccine candidate can be generated. In vitro, we observed an efficient amplification of two TR-RNAs, encoding for the CHIKV and the RRV envelope proteins, by the replicase, which resulted in a high antigen expression. Vaccination of BALB/c mice with the two TR-RNAs induced CHIKV- and RRV-specific humoral and cellular immune responses. However, antibody titers and neutralization capacity were higher after immunization with a single TR-RNA. In contrast, alphavirus-specific T cell responses were equally potent after the bivalent vaccination. These data show the proof-of-principle that the taRNA system can be used to generate multivalent vaccines; however, further optimizations will be needed for clinical application.

The arthritogenic alphavirus, chikungunya virus (CHIKV), is now present in almost 100 countries worldwide. Further spread is very likely, which raises public health concerns. CHIKV infections cause fever and arthralgia, which can be debilitating and last for years. Here, we describe a CHIKV vaccine candidate based on trans -amplifying RNA (taRNA). The vaccine candidate consists of two RNAs: a non-replicating mRNA encoding for the CHIKV nonstructural proteins, forming the replicase complex and a trans -replicon (TR) RNA encoding the CHIKV envelope proteins. The TR-RNA can be amplified by the replicase in trans , and small RNA amounts can induce a potent immune response. The TR-RNA was efficiently amplified by the CHIKV replicase in vitro , leading to high protein expression, comparable to that generated by a CHIKV infection. In addition, the taRNA system did not recombine to replication-competent CHIKV. Using a prime-boost schedule, the vaccine candidate induced potent CHIKV-specific humoral and cellular immune responses in vivo in a mouse model. Notably, mice were protected against a high-dose CHIKV challenge infection with two vaccine doses of only 1.5 μg RNA. Therefore, taRNAs are a promising safe and efficient vaccination strategy against CHIKV infections., Competing Interests: U.S., T.B., and M.P. are inventors on patents and patent applications that cover parts of this article. U.S. is an employee at BioNTech Corporation (Mainz, Germany), a privately owned company developing therapeutic RNA. All others declare no competing interests., (© 2022 The Authors.)

PLAC1 (placenta enriched 1) is a mammalian trophoblast-specific protein. Aberrant expression of PLAC1 is observed in various human cancers, where it is involved in the motility, migration, and invasion of tumor cells, which are associated with the phosphoinositide 3-kinase (PI3K)/AKT pathway. We previously demonstrated that AKT activation mediates the downstream effects of PLAC1; however, the molecular mechanisms of PLAC1-induced AKT-mediated tumor-related processes are unclear. We studied human choriocarcinoma and breast cancer cell lines to explore the localization and receptor-ligand interactions, as well as the downstream effects of PLAC1. We show secretion and adherence of PLAC1 to the extracellular matrix, where it forms a trimeric complex with fibroblast growth factor 7 (FGF7) and its receptor, FGF receptor 2 IIIb (FGFR2IIIb). We further show that PLAC1 signaling via FGFR2IIIb activates AKT phosphorylation in cancer cell lines. As the FGF pathway is of major interest in anticancer therapeutic strategies, these data further promote PLAC1 as a promising anticancer drug target., Competing Interests: CONFLICTS OF INTEREST OT was the founder and chief executive officer of Ganymed until the end of 2016, and is currently an employee and chief medical officer of BionTech. In addition, OT holds several patents for the investigational agent, zolbetuximab, with royalties paid to Astellas, and has also received consultancy fees from Astellas Pharma. US was the co-founder and shareholder at Ganymed and also holds several patents, with royalties paid to Astellas. He is currently the founder, chief executive officer, and shareholder of BioNTech. DBR, MG, SH-R, C Hartmann MK, hold a patent EP 2 662 386 A1 licensed to BioNTech AG, TRON gGmbH, Universitätsmedizin der Johannes Gutenberg-Universität Mainz. CR was a former employee of Ganymed. GC, TB, and SW have no conflicts of interest to disclose.

Here, we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10- to 100-fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- over saRNA-encoded replicase to drive expression of the transreplicon is most likely attributable to its higher translational efficiency and lack of interference with cellular translation. Testing the novel taRNA system in mice, we observed that doses of influenza hemagglutinin antigen-encoding RNA as low as 50 ng were sufficient to induce neutralizing antibodies and mount a protective immune response against live virus challenge. These findings, together with a favorable safety profile, a simpler production process, and the universal applicability associated with this bipartite vector system, warrant further exploration of taRNA., (Copyright © 2019. Published by Elsevier Inc.)

Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3' UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3' UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) the delivery of vaccine antigens in order to mount T cell immune responses and (2) the introduction of reprogramming factors into differentiated cells in order to induce pluripotency, that mRNAs tagged with the 3' UTR elements discovered in this study outperform those with commonly used 3' UTRs. This approach further leverages the utility of mRNA as a gene therapy drug format., (Copyright © 2018. Published by Elsevier Inc.)

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 μg sa-RNA compared to 80 μg mRNA (64-fold less material). Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Recombinant vaccine strain-derived measles virus (MV) is clinically tested both as vaccine platform to protect against other pathogens and as oncolytic virus for tumor treatment. To investigate the potential synergism in anti-tumoral efficacy of oncolytic and vaccine properties, we chose Ovalbumin and an ideal tumor antigen, claudin-6, for pre-clinical proof of concept. To enhance immunogenicity, both antigens were presented by retroviral virus-like particle produced in situ during MV-infection. All recombinant MV revealed normal growths, genetic stability, and proper expression and presentation of both antigens. Potent antigen-specific humoral and cellular immunity were found in immunized MV-susceptible IFNAR -/- -CD46Ge mice. These immune responses significantly inhibited metastasis formation or increased therapeutic efficacy compared to control MV in respective novel in vivo tumor models using syngeneic B16-hCD46/mCLDN6 murine melanoma cells. These data indicate the potential of MV to trigger selected tumor antigen-specific immune responses on top of direct tumor lysis for enhanced efficacy.

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.

Unlabelled: In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR(-/-))-CD46Ge mice with 2 × 10(5) 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice., Importance: Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The BCR/ABL oncogene is responsible for the phenotype of Philadelphia chromosome-positive (Ph+) leukemia. BCR/ABL exhibits an aberrant ABL-tyrosine kinase activity. The treatment of advanced Ph+ leukemia with selective ABL-kinase inhibitors such as Imatinib, Nilotinib and Dasatinib is initially effective but rapidly followed by resistance mainly because of specific mutations in BCR/ABL. Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation reduces its kinase activity and increases the sensitivity for Imatinib. We show that (i) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both unmutated and mutated BCR/ABL; (ii) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; and (iii) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings support earlier observations that BCR/ABL harboring the T315I mutation have a transformation potential that is at least partially independent of its kinase activity. These data provide evidence that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance., ((c) 2008 Wiley-Liss, Inc.)

Background: The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome--Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR., Methods: We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1alpha gradient., Results: Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells., Conclusion: Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.

Acute myeloid leukemia is characterized by a differentiation block as well as by an increased self-renewal of hematopoietic precursors in the bone marrow. This phenotype is induced by specific acute myeloid leukemia-associated translocations, such as t(15;17) and t(11;17), which involve an identical portion of the retinoic acid receptor alpha (RARalpha) and either the promyelocytic leukemia (PML) or promyelocytic zinc finger (PLZF) genes, respectively. The resulting fusion proteins form high molecular weight complexes and aberrantly bind several histone deacetylase-recruiting nuclear corepressor complexes. The amino-terminal BTB/POZ domain is indispensable for the capacity of PLZF to form high molecular weight complexes. Here, we studied the role of dimerization and binding to histone deacetylase-recruiting nuclear corepressor complexes for the induction of the leukemic phenotype by PLZF/RARalpha and we show that (a) the BTB/POZ domain mediates the oligomerization of PLZF/RARalpha; (b) mutations that inhibit dimerization of PLZF do the same in PLZF/RARalpha; (c) the PLZF/RARalpha-related block of differentiation requires an intact BTB/POZ domain; (d) the mutations interfering with either folding of the BTB/POZ domain or with its charged pocket prevent the self-renewal of PLZF/RARalpha-positive hematopoietic stem cells. Taken together, these data provide evidence that the dimerization capacity and the formation of a functionally charged pocket are indispensable for the PLZF/RARalpha-induced leukemogenesis.

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts, an effect achieved either alone or in combination with differentiating agents, such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore, valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4, a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary, we here show that valproic acid, known to induce differentiation or apoptosis in leukemic blasts, stimulates the proliferation of normal HSC, an effect with a potential effect on its future role in the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the block of differentiation, deregulated apoptosis, and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha), promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha), and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore, we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference, and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition, the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML.

Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

Since the 19th century, arsenic (As2O3) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As2O3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As2O3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARalpha or BCR/ABL. The PML portion of PML/RARalpha is crucial for the sensitivity to As2O3. PML is nearly entirely contained in PML/RARalpha. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ABL could increase sensitivity to As2O2-induced apoptosis by modifying PML expression. To disclose the mechanism of As2O3-induced apoptosis in PML/RARalpha- and BCR/ABL-expressing cells, we focused on the role of PML for As2O3-induced cell death. Here we report that (i) sensitivity to As2O3-induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As2O3-induced apoptosis; (iii) both BCR/ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon alpha-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As2O2-induced cell death.

Acute myeloid leukemia (AML)-associated chromosomal translocations result in formation of chimeric transcription factors, such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, of which the components are involved in regulation of transcription by chromatin modeling through histone acetylation/deacetylation. The leukemic differentiation block is attributed to deregulated transcription caused by these chimeric fusion proteins, which aberrantly recruit histone-deacetylase (HDAC) activity. One essential differentiation pathway blocked by the leukemic fusion proteins is the vitamin (Vit) D(3) signaling. Here we investigated the mechanisms by which the leukemic fusion proteins interfere with VitD(3)-induced differentiation. The VitD(3)-receptor (VDR) is, like the retinoid receptors RAR, retinoid X receptor, and the thyroid hormone receptor (TR), a ligand-inducible transcription factor. In the absence of ligand, the transcriptional activity of TR and RAR is silenced by recruitment of HDAC activity through binding to corepressors. In the presence of ligand, TR and RAR activate transcription by releasing HDAC activity and by recruiting histone-acetyltransferase activity. Here we report that VDR binds corepressors in a ligand-dependent manner and that inhibition of HDAC activity increases VitD(3) sensitivity of HL-60 cells. Nevertheless, the inhibition of HDAC activity is unable to overcome the block of VitD(3)-induced differentiation caused by PLZF/RARalpha expression. Here we demonstrate that the expression of the translocation products PML/RARalpha and PLZF/RARalpha impairs the localization of VDR in the nucleus by binding to VDR. Furthermore, the overexpression of VDR in U937 cells expressing AML-related translocation products completely abolishes the block of VitD(3)-induced differentiation. Taken together these data indicate that the AML-associated translocation products block differentiation not only by interfering with chromatin-modeling but also by sequestering factors involved in the differentiation signaling pathways, such as VDR in the VitD(3)-induced differentiation.

The IL-1beta gene is rapidly and transiently expressed in LPS-stimulated macrophages. While several studies have addressed the molecular basis of LPS-induced transcriptional activity, the mechanisms which underlie the subsequent decrease in IL-1beta gene expression have not been as extensively examined. In this regard, we found that the characteristic decrease in IL-1beta production after LPS stimulation could be abrogated by treatment of macrophages with the protein kinase inhibitor staurosporine. This inhibitor mediated an enhancement of IL-1beta production which was first evident 8-12 h after LPS stimulation and continued at peak levels for the rest of the incubation period (24 h). IL-1beta production was correlated with the level of mRNA specific for the cytokine. Staurosporine also mediated an enhancement of LPS-induced IL-1beta promoter activity measured in RAW 264.7 cells transiently transfected with an IL-1beta reporter plasmid. This increase paralleled the enhancement of IL-1beta mRNA by staurosporine both in intensity and time after LPS stimulation, suggesting that the negative regulation of IL-1beta is exerted primarily at the level of transcription. This regulation may be at least partially due to an observed inhibition of nitric oxide production by staurosporine in LPS-activated macrophages, which was correlated with enhanced IL-1beta production. However, the intensity of the observed effects suggested that additional staurosporine-sensitive regulatory mechanisms are in operation at the level of promoter activity., (Copyright 2001 Academic Press.)