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- The role of oncogenic Cbl mutants in Kit signaling and myeloid transformation (2009)
- Acute myeloid leukemia (AML) is a hematopoietic cell disorder characterized by a block in differentiation and increased proliferation and survival of malignant blasts. Expansion of the malignant cell clone effects the normal production of blood cells and – if left untreated – leads to death. Receptor tyrosine kinases (RTKs) play an important role in the pathogenesis of AML, as they are either often mutated or overexpressed. In normal hematopoiesis, RTK signal termination is tightly controlled, and involves ubiquitination, internalization, endocytosis and degradation. Cbl proteins are E3 ligases and have been shown to ubiquitinate several activated RTKs, including Flt3 and Kit, targeting them for degradation. Recently, several Cbl mutations have been identified: Cbl-R420Q was identified in an AML patient and Cbl-70Z was identified in a mouse lymphoma model. In this thesis work, the role of these Cbl mutants in Kit signaling and in a mouse transplantation model was studied. Cbl mutants (Cbl-R420Q, Cbl-70Z) have the ability to transform the myeloid 32D cell line in cooperation with Kit WT. Cbl mutants along with Kit promoted interleukin-3 (IL3)-independent proliferation and enhanced the cell survival of 32D cells. In contrast, expression of the Cbl mutants alone did not confer IL3-independent growth. Stem cell factor (SCF, the Kit ligand) dependent growth was enhanced in the presence of Cbl mutants and Cbl mutants promoted colonogenic growth in the presence of Kit. Furthermore, Cbl mutants inhibited the ubiquitination of the activated Kit receptor. In addition, Cbl mutants inhibited the endocytosis of the activated Kit receptor. Retroviral expression of Cbl mutants in transplanted bone marrow induced a generalized mastocytosis, a myeloproliferative disease and, in rare care cases, myeloid leukemia. Splenomegaly was observed in the presence of Cbl mutants. Furthermore, mast cells with variable range of infiltration were noticed in all the vital organs (spleen, liver, bone marrow, lung, kidney, heart) of Cbl (mutant) transplanted mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. This is the first published report on a hematological disease with Cbl mutants in a mouse model. Co-immunoprecipitation studies indicated that Cbl-70Z binds to Kit, even in the absence of Kit ligand. Cbl-R420Q also bound to Kit in the absence of SCF, albeit to a lesser extent. Association of Cbl mutants to Kit was enhanced in the presence of SCF. Signaling studies demonstrated the constitutive activation of Akt and Erk in the presence of Cbl mutants and Kit. In addition, Cbl mutants enhanced the SCF-dependent Kit, Akt and Erk activation. Cbl-70Z, in association with kinase-dead Kit (Kit-KD) or kinase-dead Flt3 (Flt3-KD), conferred IL3-independent growth and survival to the myeloid 32D cell line. Cbl-R420Q provided only a slight growth advantage in the presence of Kit-KD. As demonstrated by pharmacological inhibition studies, Akt activation was necessary for the transformation mediated by Cbl-70Z and Kit-KD / Flt3-KD. Cbl mutants enhanced the Src family kinases (SFKs) activity. The pharmacological inhibition of SFK activity inhibited the proliferation and colonogenic growth. Interaction was found between Cbl-70Z, SFKs and Kit-KD. The SFK member Fyn was identified to bind to Cbl. In addition, kinase activity of SFKs was necessary for binding to Cbl, since SFKs inhibition by PP-2 abolished the binding between the complex-binding partners. Dasatinib and PP-2, both SFK inhibitors, inhibited the Cbl and Akt phosphorylation indicating that Fyn acts upstream of Akt. Inhibition of Kit with imatinib reduced the proliferation of cells overexpressing Kit WT and Cbl-70Z much stronger compared with cells expressing Kit-KD and Cbl-70Z, but much less than the dual KIT/SFK inhibitor dasatinib. This indicated that Kit kinase activity was required but not essential. The data presented in this thesis work implies that both RTK and SFK inhibition may have to be targeted, in order to effectively prevent transformation. In summary, the present thesis work indicates an important role of Cbl, Kit and SFKs in myeloid transformation and deregulated signal transduction.
- Humanized mice as preclinical model for HIV infections (2014)
- HIV vaccine preclinical testing is difficult because HIV’s only relevant hosts are humans and no correlates of protection are known. To this end, we are working on the humanization of different mouse strains with human peripheral blood mononuclear cells (PBMCs) as well as human hematopoietic stem cells (HSC) to generate a useful small animal model. We generated immune deficient mice (NOD Scid IL2gc -/- /NOD Rag1-/- IL2gc -/-) expressing human MHC class II (HLA-DQ8) on a mouse class II deficient background (Ab-/-). Here, the human HLA-DQ8 should interact with the matching T cell receptors of transferred matching human PBMCs and therefore could support the functionality of the transferred human CD4+ cells in the mice. Mice that were adoptively transferred with human HLA-DQ8 PBMCs only showed engraftment of CD3+ T cells. Surprisingly, the presence of HLA class II did not significantly change the repopulation rates in the mice. Also, the presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was significantly prolonged, compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GvHD. To avoid GVHD and to increase and maintain the level of human cell reconstitution over a long period of time, the same mouse strains were reconstituted with human HSC. Compared to PBMC-repopulated mice, HSC-reconstituted mice develop almost all subpopulations of the human immune system detectable at week 12 after HSC transfer. These mice developed adaptive immune responses after Tetanus Toxoide (TT) immunizations. In addition, we are testing the susceptibility of these humanized mice to different HIV strains with a detailed look at immune responses.
- T cell receptor diversity prevents T cell leukemia, lymphoma development / von Nabil Saleh Ahmed Al-Ghaili (2010)
- Gene therapy is a promising therapeutic strategy that emerged from the attractive idea of targeting therapy at the molecular level. For many patients who suffer from genetic and acquired diseases that cannot be effectively treated by conventional treatment approaches gene therapy remains a huge hope of cure in spite of the hurdles regarding efficacy and safety that need to be overcome. The development of efficient gene transfer vehicles, mainly retroviral vectors, led to the first successful gene therapy trial, to treat patients suffering from X-linked severe combined immunodeficiency syndrome (X-SCID) using gene modified stem cells (Hacein-Bey-Abina, Le Deist et al. 2002). Despite the success of this trial, it revealed the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy using gene-modified stem cells (Hacein-Bey-Abina, von Kalle et al. 2003). In contrast to stem cells, T cells are relatively resistant to insertional mutagenesis and transformation even after transduction with potent oncogenes using retroviral vectors (Newrzela, Cornils et al. 2008). However, mature T cells can self-renew, proliferate and survive for long periods. These criteria are supposed to render T cells prone to transformation. Therefore, the questions of mature T cells transformability and the control mechanism limiting their transformation are still elusive.
- Effect of chromatin modeling by histone deacetylase inhibitors (HDIs) on hematopoietic stem cell (HSC) fate (2005)
- Acute myeloid leukemia (AML) is characterized by the accumulation of a large number of abnormal, immature blast cells. Recently, histone deacetylase inhibitors (HDIs) received considerable interest on the ground of their ability to overcome the differentiation block in these leukemic blasts regardless of the primary genetic alteration, an effect achieved either alone or in combination with differentiating agents, such as all-trans retinoic acid (t-RA). Valproic acid (VPA), a potent HDI, is now under clinical evaluation owing to its potent differentiation effect on transformed hematopoietic progenitor cells and leukemic blasts from AML patients. Conversely, in a clinical study by Bug et al., the favorable effects of the combination treatment with t-RA/VPA in advanced acute myeloid leukemia patients were reported to be most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. Based on the hypothesis that VPA influences normal hematopoiesis, the effect of chromatin modeling through VPA on HSCs was investigated with respect to differentiation, proliferation as well as self-renewal in the present study. It has been shown that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21cip-1/waf-1. Furthermore, valproic acid inhibits GSK3B 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. To sum up, valproic acid, a potent histone deacetylase inhibitor known to induce differentiation and/or apoptosis in leukemic blasts, stimulates the proliferation and self-renewal of hematopoietic stem cells. Therefore, the data reported in this study suggest to reconsider the role of histone deacetylase inhibitors from a differentiation inducer to a coadjuvant factor for increasing the response to conventional therapy in acute myeloid leukemia.
- Role of rho GTPases in migration of stem and progenitor cells (2005)
- Stem cells capable of self-renewal and differentiation into multiple tissues are important in medicine to reconstitute the hematopoietic system after myelo-ablative chemo- or radiotherapy. In the present situation, adult stem cells such as Mesenchymal stem cells (MSC) and Hematopoietic stem cells (HSC) are used for therapeutic purposes. For tissue regeneration and tissue constitution, engraftment of transplanted stem cells is a necessary feature. However, in many instances, the transplanted stem cells reach the tissues with low efficiency. Considering the three-step model of leukocyte extravasation by Springer et al, the rolling, adhesion and transmigration form the three major steps for the transplanted stem cells to enter the desired tissues. One of the molecular switches reported to be involved in these mechanisms are the Rho family GTPases. The present study investigates the role of Rho GTPases in adhesion and migration of stem and progenitor cells. Chemotactic and chemokinetic migration assays, transendothelial migration assays, migration of cells under shear stress, microinjection, retroviral and lentiviral gene transfer methods, oligonucleotide microarray analysis and pull down assays were employed in this study for the elucidation of Rho GTPase involvement in migration and adhesion of stem and progenitor cells. The transmigration assay used for the migration determination of the adherent cell type, MSC, was optimized for the efficient and effective assessment of the migrating cells. The involvement of Rho was found to be critical for stem and progenitor cell migration where inactivation of Rho by C2I-C3 transferase toxin and/or overexpression of C3 transferase cDNA increased the migration rate of Hematopoietic progenitor cells (HPC) and MSC. Moreover, modulation of Rho caused predictable cytoskeletal and morphological changes in MSC. Assessment of Rho GTPase involvement in the interacting partner, the endothelial cells during stem cell migration, revealed that active Rho expression induced E-selectin expression. The increased levels of E-selectin were functionally confirmed by the increased adhesion of progenitor cells (HPC) to the Human umbilical vein endothelial cell (HUVEC) layer. Moreover, inhibition of Rac in the migrating endothelial progenitor cells (eEPC) increased their adhesion to HUVEC correlating with the increased percentage expression of cell surface receptor, CD44 in Rac inactivated eEPC. In conclusion, this study shows that Rho GTPases control the adhesion and migration of stem and progenitor cells, HPC and MSC. Rho inhibition drives the cells to migrate in the blood vessels. The substantial increase in the level of active Rho in endothelial layer, manifested by the E-selectin surface expression assists the better adhesion of stem and progenitor cells to the endothelial layer. Serum factors and growth factors in the physiological system influence the Rho GTPase expression in both migrating stem cells and the barrier endothelial cells. Thus, specific modulation of Rho GTPases in the transplanted stem and progenitor cells could be an interesting tool to improve the migration and homing processes of stem cells for cellular therapy in future.
- Untersuchungen zur Bedeutung des Kern-Zytoplasma Transports für die biologische Funktion zellulärer Proteine (2005)
- The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
- Retargeted natural killer cells for adoptive cancer immunotherapy (2011)
- NK cells are part of the innate immune system, and are important players in the body’s first defence line against virus-infected and malignantly transformed cells. While T cells recognize neoplastic cells in an MHC-restricted fashion, NK cells do not require prior sensitization and education about the target. In leukemia and lymphoma patients undergoing allogeneic hematopoietic stem cell transplantation not only T cells but also NK cells have been found to mediate potent graft-versus-tumor effects. Hence, autologous or donor-derived NK cells hold great promise for cancer immunotherapy. Since the generation of highly purified NK cell products for clinical applications is labor-intensive and time consuming, established human NK cell lines such as NK-92 are also being considered for clinical protocols. NK-92 cells display phenotypic and functional characteristics similar to activated primary NK cells. While NK-92 cells are highly cytotoxic towards malignant cells of hematologic origin, they do not affect healthy human tissues. NK-92 cells can be expanded under GMP-compliant conditions, and can therefore be provided in sufficient numbers with defined phenotypic characteristics for clinical applications. Safety of NK-92 cells for adoptive immunotherapy was already shown in two phase I/II clinical trials. In contrast to malignant cells of hematologic origin, most solid tumor cells are not sensitive to unmodified NK-92 cells. Hence, to overcome resistance mechanisms of tumor cells and to broaden the target spectrum of NK-92 cells, gene-modified variants have been generated which express chimeric antigen receptors (CARs) that specifically target tumor surface antigens. The expression of these CARs is sufficient to redirect their cytotoxic activity towards otherwise NK cell-resistant target cells. Extending these earlier approaches, in the framework of this work optimized CAR constructs that target the pancarcinoma antigen epithelial cell adhesion molecule (EpCAM) were derived and functionally characterized. In collaboration with Heike Daldrup-Link’s laboratory (University of California San Francisco, USA) non-invasive imaging modalities to analyze biodistribution and tumor homing properties of retargeted NK-92 cells were evaluated. To enhance the persistence of adoptively transferred NK-92 cells in vivo, means to overcome NK-92 cells’ dependence on exogenous IL-2 for survival and cytolytic activity were investigated. EpCAM is expressed on a variety of tumors of epithelial origin including ovarian, gastric, colorectal, pancreatic, breast, lung and endometrial cancers. In epithelial cells EpCAM is mainly expressed at basolateral membranes, and EpCAM is involved in calcium-independent homotypic cell-cell adhesions. In tumor cells high and de novo EpCAM expression is not only restricted to basolateral membranes but can also be found on apical membranes. Tumor cells retain EpCAM expression throughout tumorigenesis and metastasis formation. Due to its surface expression and immunogenicity EpCAM has been exploited as target for immunotherapy. In earlier work in our group a prototypic, first generation EpCAM-specific CAR construct (31.z) harboring a murine flexible hinge region and murine CD3 ζ as signaling domain was derived and functionally characterized in NK-92 cells. To reduce the immunogenicity for their potential clinical application, this CAR construct was humanized by exchanging the hinge region and the intracellular signaling domain with corresponding sequences of human origin. In T cells incorporation of additional co-stimulatory domains derived from CD28 and 4-1BB significantly enhanced persistence and anti-tumor effects of adoptively transferred cells. Based on these findings a modified, second generation CAR construct encompassing transmembrane and intracellular regions of CD28 in addition to CD3 ζ intracellular signaling domains was derived (31.28.z). Both CAR constructs were stably expressed in NK-92 cells, and furthermore, expression of both CAR variants promoted antigen-specific lysis of antigen-expressing prostate and breast cancer cell lines. In competition experiments the cytotoxic activity of NK-92/31.z and NK-92/31.28.z cells towards antigen-expressing tumor cells was significantly reduced in the presence of parental MOC31 monoclonal antibody, indicating that binding of the EpCAM-specific CAR to its antigen on tumor cells is necessary to trigger antigen-specific cytotoxicity. At high effector to target ratios NK-92/31.28.z cells displayed slightly higher cytotoxic activity towards EpCAM-expressing target cell lines than NK-92/31.z cells, suggesting that incorporation of co-stimulatory domains had beneficial effects on the cytotoxic activity. For clinical applications the development of non-invasive imaging methods is necessary to follow the biodistribution of adoptively transferred cells and guide the identification of responders and non-responders at an early time point. In collaboration with Heike Daldrup-Link’s laboratory the homing properties of EpCAM-specific NK-92 cells to prostate tumor xenografts in rodent models was analyzed (University of California San Francisco, USA). At that time NK-92 cells expressing the second generation EpCAM-specific CAR 31.28.z were not yet available, and thus homing experiments were performed with NK-92 cells expressing the first generation CAR 31.z. For magnetic resonance imaging studies parental and EpCAM-specific NK-92 cells were labeled with clinical applicable ferumoxide particles. Labeled, gene-modified NK-92 cells displayed reduced CAR expression and reduced cytotoxic activity towards EpCAM-expressing DU145 prostate cancer cells in vitro. Nevertheless, MRI revealed specific accumulation of ferumoxide labeled EpCAM-specific NK-92 cells in DU145 tumor xenografts in athymic rats. In tumor sections of treated animals the presence of EpCAM-specific NK-92 cells was verified by Prussian blue and CD57 staining of tumor sections. In another study homing of DiD-labeled EpCAM-specific NK-92 cells to DU145 tumor xenografts was shown by optical imaging. These findings imply that specific targeting of NK-92 cells is retained in vivo, and that non-invasive imaging strategies can be employed to analyze biodistribution of NK-92 cells. Enhanced persistence of adoptively transferred cytotoxic effector cells has a major impact on the effectiveness of immunotherapy. Primary cytotoxic effector cells as well as NK-92 cells require IL-2 for their proliferation and to gain full activity of their effector functions. To bypass the need of exogenously supplied cytokines, the expression of chimeric cytokine receptors (CCR) harboring IL-2R β and IL-2R γ chains instead of CD3 ζ as signaling domains might initiate cytokine-like signals upon contact with the respective antigen. These interactions might support growth and survival of NK-92 cells in the absence of exogenous IL-2. As a starting point, a codon-optimized ErbB2-specific CAR consisting of the scFv(FRP5) single chain antibody fragment, a human CD8 α hinge region and human CD3 ζ transmembrane and intracellular domains was used. Transmembrane and intracellular domains of IL-2R β and IL-2R γ chains were amplified from NK-92 cell-derived cDNA, and were used to exchange the CD3 ζ domain in the ErbB2-specific construct. In human primary tumors EpCAM and ErbB2 overexpression are frequently found, and often correlate with poor prognosis. Hence, co-expression of ErbB2-specific CCRs with an EpCAM-specific CAR may provide NK cells with antigen-specific killing via EpCAM recognition and with antigen-dependent growth via binding to ErbB2. However, attempts to activate CCRs in NK-92 cells via co-incubation with antigen-expressing cells or cross-linking of the CCRs with recombinant antigen did not result in cytokine-independent but antigen-dependent growth. Likewise, no triggering of signal transducer and activator of transcription 5 (STAT5) was observed, which is a hallmark of IL-2 mediated signal transduction. The interactions between CCRs and their antigen might not be strong enough to trigger cytokine-like signals supporting the growth of cells in the absence of exogenous cytokines, and furthermore, might not lead to a significant up-regulation of STAT5-mediated signal transduction. An alternative approach to circumvent the need of exogenous cytokines is ectopic expression of homeostatic cytokines IL-2 and IL-15 in lymphocytes. In T cells expression of these cytokines is sufficient to render cells independent from exogenously supplied cytokines. In this work a lentiviral expression vector encoding IL-15 (SIEW-IL15) was generated, and used for transduction of NK-92 cells. This resulted in ectopic expression of IL-15 and cellular proliferation in the absence of exogenously supplied cytokines. Even after prolonged culture without exogenous IL-2, NK-92/IL15 cells retained their cytotoxic activity towards NK-sensitive target cells. Although expression of IL-15 in HC11 and COS-7 cells using the same vector led to secretion of bioactive IL-15 into culture supernatants, neither secreted nor surface-bound IL-15 was detected in NK-92/IL15 cells, implying that IL-15 promotes survival of gene-modified cells in a strictly autocrine fashion. In addition, NK-92 cells that were freshly transduced with SIEW-IL15 could be efficiently enriched by cytokine withdrawal. NK-92/IL15 cells that were co-transduced with an EpCAM-specific CAR retained their ability to grow in the absence of exogenously supplied cytokines and their antigen-specific cytotoxic activity. Based on these results, a bicistronic vector construct was generated allowing the simultaneous expression of a CAR construct and IL-15 as selection marker. EpCAM-specific CAR constructs (31.28.z and 31.TM) were inserted into the bicistronic expression cassette. NK-92 cells were transduced with these bicistronic expression constructs and selected by cytokine withdrawal. After 14 to 21 days of culture in the absence of IL-2 transduced cells grew out from which CAR-expressing NK-92 cells with high and homogenous surface expression were further enriched by FACS sorting. NK-92/31.28.z.IL15 cells displayed high cytotoxic activity towards EpCAM-expressing breast cancer cell lines, while EpCAM-negative melanoma cells were not lysed. The results of this work demonstrate that the expression of first (31.z) and second (31.28.z) generation CARs in NK-92 cells is sufficient to induce antigen-specific cytotoxicity. Furthermore, a specific accumulation of NK-92/31.z cells but not unmodified NK-92 cells was detected in EpCAM-expressing prostate carcinoma xenografts in athymic rats, indicating that specific targeting of these cells is retained in vivo. Ectopic expression of IL-15 renders the cells independent from exogenous cytokines, while they retain their cytotoxic activity even after prolonged culture without IL-2. Furthermore, ectopic expression of IL-15 in NK-92 cells can be used for selective enrichment of gene-modified cells by cytokine withdrawal. Subsequently, bicistronic expression constructs that allow simultaneous expression of a CAR construct and IL-15 as selection marker were generated. Expression of these bicistronic expression vectors in NK-92 cells is feasible, and might facilitate enrichment of gene-modified cells for clinical applications.
- Novel approaches of molecular targeting in Philadelphia chromosome positive leukemia (2009)
- In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/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. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; 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 suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.
- Development of lentiviral vectors for the gene therapy of HIV infection (2010)
- Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer” tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10 HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral combination therapy. T cell infusions were tolerated well with no severe side effects. A significant increase of CD4 counts was observed post infusion. At the end of the one-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear follow-up, whereby marking levels correlated with the cell dose. No significant changes of viral load were observed during the first four months. Four of the seven patients that changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking and may improve immune competence in HIV-infected patients with advanced disease and multidrug resistant virus. However, the low level of gene marking and the lack of substantial long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate the requirement for new vectors with new strategy. In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from this work provide the basis for the selection of a suitable candidate vector for extensive preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors incorporate a number of additional features that are of potential value for gene therapeutic applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration profile. The use of internal promoters to drive expression of the therapeutic transgene maC46 should further improve the safety profile of these new‐generation vectors, while an additional artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene expression. The rationale for this is that hematopoietic stem and progenitor cells will be Summary 98 protected from enhancer‐mediated transactivation effects and also from potential side effects due to the aberrant expression of maC46 while at the same time the full clinical benefit for the patients is maintained. In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors harboring different regulatory elements were selected based on results from pilot experiments. The internal promoters used to drive expression of codon optimized maC46 were the PGK promoter and MPSV promoter. This work focuses on the transgene expression levels in lymphoid cells and antiviral activity. The issues of long term expression, propensity to methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first step the performance of different vectors was evaluated in the human T cell lines. Based on promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the MPSV promoter along with intron were selected for in vivo transplantation experiments. In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells, along with maintained expression of introduced genes. It was observed that the expression of these constructs depends strongly on the activation and differentiation status of the targeted T cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46 can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. . In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of T cell activation status as related to the desired clinical effect must be addressed. These results might open the way for a gene therapy targeting mainly or exclusively activated T cells and could be exploited for immunostimulatory as well as suppressive approaches.
- Method developments in coupling gel electrophoresis with mass spectrometry (2011)
- Proteomic analysis is the large-scale identification and characterization of proteins including post translational modifications. Proteomics encompasses a number of approaches including bottom-up and top-down workflows which are widely used independently and complementary as tools for the successful study of protein species. However, up to the present day these techniques have not been able to overcome every analytical limitation. Mass spectrometry has played a vital role alongside proteomics in providing the required analytical means of detecting protein amounts down to the atomole range. Soft ionization methods such as matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have permitted the transfer of peptides and intact proteins into the gas phase without extensive degradation. The introduction of recent developments in MALDI technology such as the highly sensitive 4-chloro-alpha-cyanocinnamic acid matrix (Cl-CCA) as well as the commercial availability of a MALDI-LTQ-Orbitrap which boosts peptide mass accuracy below 3 parts per million (ppm), have offered new prospective in protein analysis. The aim of the current study is to incorporate these new aspects and provide further advancements in gel-based as well as gel-free proteomic workflows. Peptides of proteolytically digested proteins are routinely analyzed by means of peptide mass fingerprinting (PMF) often combined with MS/MS analyses to complement and substantiate PMF results by peptide sequence information. The most widely used protease for enzymatic digestion is trypsin, since it exhibits a very specific cleavage behavior limited to C-terminal hydrolyses after basic amino acids. However, less specific enzymes such as chymotrypsin, elastase and pepsin have emerged as useful tools in the analysis of particular protein classes e.g. membrane, cereal, and phosphorylated proteins. In this work a comprehensive bottom-up proteomic investigation including in-solution and in-gel protein digestions of analytes covering small to large, acidic to basic, and hydrophobic to hydrophilic proteins in combination with a series of less specific enzymes are presented in order to show the superiority of the novel MALDI matrix Cl-CCA. The Cl-CCA matrix proved to be highly superior compared to standard α-cyano-4-hydroxycinnamic acid (CHCA) since an average detection of more than 2- to 3-fold peptide amount was possible depending on the used protease and, therefore, resulting in strongly increased sequence coverage. Additionally, protein identification of chymotrypsin and elastase in-gel digested protein standards was evaluated. The MALDI-LTQ-Orbitrap providing peptide mass accuracy below and up to 3 ppm in combination with Cl-CCA as matrix and newly optimized digestion conditions led to unambiguous protein identifications of all chymotryptic digests outperforming its tryptic counterparts in the case of hydrophobic bacteriorhodopsin and α-globin from hemoglobin A (α-HgbA). In addition, significantly higher sequence coverage and increased number of detected peptides was acquired. Moreover, a proposed workaround for elastase digestions was capable of providing a solution for successful identification results. Apart from digestions of singly separated proteins, solution isoelectic focusing (sIEF) was evaluated. OFFGEL fractionation is an efficient means of fractionating peptides and proteins according to their isoelectric point (pI) values through immobilized pH gel (IPG) strips after which samples are recovered in solution. Consequently, an issue of peptide recovery arises as a category of peptides relatively insoluble to the recovery solution should be present. A method was developed including the scraping of gel matrix from the IPG strips and peptide extraction using acetonitrile as organic solvent in combination with analytical techniques such as nLC-MALDI-MS/MS for peptide identification. The nature of the peptide species remaining in-gel was analysed and attributed to peptide solubility. A general trend in which a high percentage of neutral and hydrophobic peptides remaining entrapped in the IPG gel strip was observed. The present work also examines a new top-down proteomic workflow involving protein elution from cleavable gels containing the labile crosslinker ethylene-glycol-diacrylate (EDA). Protein amounts of as low as 100 ng loaded onto EDA gels were detected using MALDI-TOF MS in the linear acquisition mode. Proteins from 8.5 up to 78 kDa were successfully measured including a hydrophobic 15 kDa core protein attaining a GRAVY score of +0.079. Additionally, the method was compatible with one dimensional protein separation as well as for 2-D IEF/SDS-PAGE. Lastly, two methods for protein identification were tested and found to be compatible to the proposed technique.