Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.

The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh-/- cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.

Resistance against tyrosine kinase inhibitors (TKIs) represents a relevant clinical problem in treatment of chronic myelogenous leukemia (CML). On the basis of their activity against the spectrum of BCR-ABL mutations that have shown to be the most prominent mechanism of resistance to imatinib, new TKIs have been classified as second generation (such as nilotinib, dasatinib and bosutinib) or third generation (also cover- ing T315I such as ponatinib) TKIs. However, mutations in BCR-ABL only account for about half of the cases of treatment failure under TKI and other mechanisms either rendering the leukemic cells still dependent of BCR-ABL activity or supporting oncogenic properties of the leukemic cells independent of BCR-ABL signaling have been identified. A detailed understanding of the different underlying resistance mechanisms will be the prerequisite to eventually overcome clinical resistance and for the successful use of tailored combinations of targeted inhibitors in the future.

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors. BIOLOGICAL SIGNIFICANCE:Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC) disease caused by the reciprocal translocation t(9;22). Although there is clear evidence that the resulting oncogenic tyrosine kinase BCR-ABL is the key event of leukemia initiation which drives stem cell proliferation and expansion of myeloid progenitors in early chronic phase (CP), the mechanism leading to advanced phases remains elusive. Recently, we could show that telomere attrition correlates with disease stages due to increased leukemic stem cell turnover. Here, we could provide first time evidence that this can functionally contribute to disease progression in CML.

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.

Secondary chromosomal aberrations may contribute to the development of a malignant phenotype in mantle cell lymphoma. Chromosomal band 5p15.33 represents a new recurrent breakpoint in B-cell malignancies. We present a molecular cytogenetic study of 8 mantle cell lymphoma (MCL) cell lines and 23 patients with MCL to determine and characterize novel secondary aberrations. We detected new secondary recurrent rearrangements in all cell lines and in 7 patients and confirmed 5p15.33 as a recurrent breakpoint in 4 cell lines and one patient. Further molecular characterization by flow-FISH and quantitative RT-PCR suggest TERT and CLPTM1L as target genes of 5p15.33 rearrangements.

Effective inhibition of BCR-ABL tyrosine kinase activity with Imatinib represents a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, more than 30 % of patients with CML in chronic phase do not respond adequately to Imatinib and the drug seems not to affect the quiescent pool of BCR-ABL positive leukemic stem and progenitor cells. Therefore, despite encouraging clinical results, Imatinib can still not be considered a curative treatment option in CML. We recently reported downregulation of eukaryotic initiation factor 5A (eIF5A) in Imatinib treated K562 cells. Furthermore, the inhibition of eIF5A by siRNA in combination with Imatinib has been shown to exert synergistic cytotoxic effects on BCR-ABL positive cell lines. Based on the structure of known deoxyhypusine synthase (DHS) inhibitors such as CNI-1493, a drug design approach was applied to develop potential compounds targeting DHS. Here we report the biological evaluation of selected novel (DHSI-15) as compared to established (CNI-1493, deoxyspergualin) DHS inhibitors. We show that upon the compounds tested, DHSI-15 and deoxyspergualin exert strongest antiproliferative effects on BCR-ABL cells including Imatinib resistant mutants. However, this effect did not seem to be restricted to BCR-ABL positive cell lines or primary cells. Both compounds are able to induce apoptosis/necrosis during long term incubation of BCR-ABL positive BA/F3 derivates. Pharmacological synergism can be observed for deoxyspergualin and Imatinib, but not for DHSI-15 and Imatinib. Finally we show that deoxyspergualin is able to inhibit proliferation of CD34+ progenitor cells from CML patients. We conclude that inhibition of deoxyhypusine synthase (DHS) can be supportive for the anti-proliferative treatment of leukemia and merits further investigation including other cancers.

3-Hydroxiglutaric acid (3-OH-GA) is a disease-specific metabolite that accumulates in tissues and body fluids of patients with Glutaric aciduria type I (GAI) and has been associated with vascular abnormalities in these kindreds. Here, we demonstrate that 3-OH-GA also affects the integrity of tumor vessels leading to tumor growth inhibition in a subcutaneous model of human glioblastoma multiforme (GBM). This effect correlated with a marked decrease of VE-Cadherin expression in endothelium of 3-OH-GA-treated tumors. Furthermore, in vitro observations indicated also a direct effect of 3-OH-GA in glioma cells that showed defective mitosis and significant proliferation inhibition. In summary, the GAI-specific metabolite 3-OH-GA significantly inhibited growth of GBM xenografts by affecting the structural integrity of tumor blood vessels and in addition by causing defective mitosis and proliferation inhibition of tumor cells.

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.

BACKGROUND:We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.RESULTS:Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.CONCLUSION:From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.

The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo.

Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34+ CD38− LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.

Targeted cancer therapies are often associated with drug resistance, a phenomenon that has been observed with tyrosine kinase inhibitors (TKIs), widely used to treat leukaemia driven by BCR–ABL1 mutations. Markus Mueschen and colleagues describe a novel BCL6-dependent mechanism of drug resistance in leukaemia through which TKI-induced upregulation of BCL6 allows leukemia cells to cope with acute oncogene withdrawal. Targeted inhibition of BCL6 reduces the number of drug-resistant and self-renewing leukaemia-initiating cells. In xenograft models of acute lymphoblastic leukaemia cells carrying BCR–ABL1 mutations, dual inhibition of BCR–ABL1 and BCL6 prevents resistance and improves the anticancer response. Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR–ABL1 (ref. 1) and other oncogenic tyrosine kinases2,3. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases4,5. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs)6,7,8, which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

Abstract Abstract 202 Background: Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months. Approach: Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53. BCL6-Null CML cells fail to initiate leukemia: Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by >15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a >12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by >7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (>400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (<5 colonies). After an initially successful engraftment of BCL6-Null CML cells in NOD/SCID mice (as visualized by luciferase bioimaging), BCL6-Null CML cells failed to initiate leukemia, whereas NOD/SCID mice injected with BCL6+/+ CML cells succumbed to the disease. Targeted inhibition of BCL6 for leukemia stem cell eradication in CML: A retro-inverso BCL6 peptide inhibitor (RI-BPI) was recently developed for targeted therapy of DLBCL. Therefore, we tested the potential therapeutic usefulness of RI-BPI for eradication of BCL6-dependent leukemia-initiating cells. Human CML cells were incubated overnight in the presence of 5 μ mol/l RI-BPI or vehicle control. While Hoechst 33342 staining revealed a distinct side population of ≂f2.8% CML cells, overnight incubation with RI-BPI reduced the frequency of ABCG2+ side population cells by more than 30-fold. Likewise, RI-BPI incubation decreased colony formation by >5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032), Conclusion: Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Disclosures: Hochhaus: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.