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Introduction: Abdominal aortic aneurysm (AAA) is a pathological saccular enlargement most often of the infrarenal aorta. Eventual rupture is fatal, making preemptive surgical therapy upon a diameter threshold of >50mm the treatment of choice. The pathophysiology, especially the initial trigger aortic remodeling is still largely unknown. However, some characteristic features involved in aneurysm growth have been established, such as medial angiogenesis, low-grade inflammation, vascular smooth muscle cell (VSMC) phenotype switch, extracellular remodeling, altered hemodynamics and an eventual humoral immune answer. Currently, no medical treatment options are available. RNA therapeutics and drug repurposing offer new possibilities to overcome this shortage. Using such to target angiogenesis in the aneurysm wall and investigate their potential mechanisms is the aim of this thesis. Material and Methods: We test our hypothesis by targeting the long non-coding RNA H19 and re-use the anti-cancer drug Lenvatinib in two murine inducible AAA models and one preclinical large animal model in the LDLR-/- pig. Furthermore, a H19-/- mouse is included to verify the results. AAA and control samples from a human biobank along with a primary human cell culture are used to verify results ex vivo by qPCR, WesternBlot, live cell imaging, histo- and immunohistochemistry along with gene array analysis, RNA knockdown, pull-down- and promotor assays. Results: H19 is significantly upregulated in AAA mice models and its knockdown limited aneurysm growth. It is well known that H19 interacts with several transcription factors. We found that cytoplasmic interaction between H19 and hypoxia-inducible factor 1-alpha (HIF1α) increased apoptosis in cultured SMCs associated with sequential p53 stabilization. In contrast, the knockdown of H19 was associated with markedly decreased apoptotic cell rates. Our data underline that HIF1α was essential in mediating the pro-apoptotic effects of H19. Secondly, Lenvatinib was applied both systemically and locally by endovascular means in mice with an established AAA. The drug significantly halted aneurysm growth and array analysis revealed myosin heavy chain 11 (MYH11) as the most differentially regulated target. This was shown to be up regulated after Lenvatinib treatment of primary AAA smooth muscle cells suggesting a salvage mechanism to obtain a contractile phenotype based on gene expression and immunohistochemistry. The same results were shown upon a local endovascular Lenvatinib-coated balloon angioplasty in the established aneurysmatic lesion of a novel atherosclerotic LDLR-/- Yucatan minipig model. Decreased phosphorylation of extracellular-signal regulated kinases 1-2 (ERK1-2) is the downstream effect of Lenvatinib-specific blockage of the vascular endothelial growth factor receptor (VEGFR2). Conclusion: Taking into account the heterogeneity of the disease, inhibition of VSMC phenotype switch, extracellular remodeling and angiogenesis seem promising targets in some if not all AAA patients. Together with surveillance and surgical therapy, these new non-invasive treatment strategies would allow for a more personalized approach to treat this disease.
Ibrutinib serves as an efficient second-line therapy in relapsed/refractory mantle cell lymphoma. However, resistance to the BTK inhibitor results in a poor prognosis for patients. Since the mechanisms leading to resistance in initially responding tumor cells are poorly understood, this work aimed to decipher acquired features in ibrutinib-surviving cells of a sensitive mantle cell lymphoma cell line and evaluate these potential therapeutic targets in ibrutinib-treated mantle cell lymphoma.
Time-resolved single-cell RNA sequencing was performed to track the transcriptomic evolution of REC-1 cells across 6 and 48 hours of treatment. Single-cell analysis uncovered a subpopulation of REC-1 with potentially greater aggressiveness and survival advantage by benefiting from interaction with the tumor microenvironment. Upregulation of B-cell receptor genes, elevated surface antigen expression of CD52 and metabolic rewiring to higher dependence on oxidative phosphorylation were identified as further potential resistance features of ibrutinib-surviving cells. RNA sequencing after prolonged incubation corroborated the increase in CD52 and oxidative phosphorylation as dominant characteristics of the cells surviving the 4-day treatment, highlighting their potential as therapeutic targets in combination with ibrutinib treatment. Concomitant use of ibrutinib and the oxidative phosphorylation inhibitor IACS-010759 increased toxicity compared to ibrutinib monotherapy due to higher apoptosis and greater inhibition of proliferation. For anti-CD52 therapy, a consecutive approach with ibrutinib pretreatment followed by incubation of surviving cells with a CD52 monoclonal antibody and human serum yielded a synergistic effect, as ibrutinib-surviving mantle cell lymphoma cells were rapidly depleted by complement-dependent cytotoxicity. Regarding the effects on primary tumor cells from mantle cell lymphoma patients, ibrutinib induced upregulation of CD52 in some cases, and increased toxicity of anti-CD52 therapy was observed in ibrutinib-sensitive patient samples after pretreatment with the BTK inhibitor. The likely favorable in vivo efficacy of an anti-CD52 therapy might therefore be restricted to a subgroup of mantle cell lymphoma patients, also in view of the associated side effects.
Given the need for new therapeutic options in mantle cell lymphoma to overcome resistance to ibrutinib, this work highlights the potentially beneficial use of an oxidative phosphorylation inhibitor as add-on therapy. In addition, the findings suggest to further assess the value of anti-CD52 therapy as consolidation to ibrutinib in ibrutinib-sensitive patients with elevated CD52 surface levels on tumor cells to target resistant clones and minimize risk of minimal residual disease and relapse.
Die WHO-Klassifikation der Hirntumoren von 2016 ebnete den Weg für molekulare Marker und Therapie-Angriffspunkte. Der Transkriptionsfaktor ATF5 könnte ein solcher sein. Er unterdrückt die Differenzierung von neuronalen Vorläuferzellen und wird in Glioblastomen (GBM) überexprimiert. Daten zur ATF5-Expression in WHO Grad II Gliomen (LGG) und GBM-Rezidiven sind nur spärlich vorhanden. Daher untersuchten wir 79 GBM, 40 LGG und 10 Normalhirnproben auf ihre ATF5-mRNA- und Proteinexpression mit quantitativer Echtzeit-PCR bzw. Immunhistochemie und verglichen sie mit multiplen, retrospektiv erhobenen klinischen Charakteristika der Patienten. ATF5 war in LGG und GBM verglichen zum Normalhirn sowohl auf mRNA-, als auch Proteinebene überexprimiert. Obwohl die ATF5-mRNA-Expression im GBM eine erhebliche Fluktuationsrate zeigte, gab es keine signifikanten Expressionsunterschiede zwischen GBM-Gruppen unterschiedlicher biologischer Wachstumsmuster. ATF5-mRNA korrelierte mit dem Alter der Patienten und invers mit der Ki67-Färbung. Kaplan Meier- und Cox-Regressionsanalysen zeigten eine signifikante Korrelation der ATF5-mRNA-Expression mit dem Überleben nach 12 Monaten sowie dem progressionsfreien Überleben. Die Methylierung des Promotors der O6-Methylguanin-DNA-Methyltransferase (MGMT) ist ein etablierter Marker in der Therapie des GBMs. Sie ist mit dem therapeutischen Ansprechen auf Temozolomid und dem Überleben assoziiert. Uns fielen inzidentell Veränderungen der MGMT-Promotormethylierung auf, woraufhin wir den aktuellen Wissensstand mittels einer ausführlichen Literatur-Metaanalyse zusammenfassten. Dabei fanden wir Veränderungen der MGMT-Promotormethylierung bei 115 der 476 Patienten. Wir schlussfolgern, dass die ATF5-mRNA-Expression als prognostischer Faktor für das Überleben der Patienten dienen könnte. Da seine in vitro-Inhibition zu einem selektiven Zelltod von Gliomzellen führte und wir eine Überexpression in glialen Tumoren nachweisen konnten, zeigt ATF5 Potential als ubiquitäres Therapieziel in Gliomen. Zum aktuellen Zeitpunkt ergibt sich keine klare Indikation, den klinischen Standard der MGMT-Teststrategie zu verändern. Trotzdem könnte eine erneute Testung der MGMT-Promotormethylierung für zukünftige Therapieentscheidungen sinnvoll sein und wir regen an, dass dieses Thema in klinischen Studien weiter untersucht wird.
Identification of new drug targets in adrenocortical carcinoma through targeted mRNA analysis
(2021)
Adrenocortical carcinomas (ACC) are aggressive tumors associated with a heterogeneous but generally poor prognosis and limited treatment options for advanced stages. Despite promising molecular insights and improved understanding of ACC biology, efficient targeted therapies have not been identified yet. Thus, this study aims to identify potential new drug targets for a future personalized therapeutic approach.
RNA was isolated from 104 formalin-fixed paraffin-embedded tumor samples from ACC patients, 40 of those 104 cases proved to be suitable for further mRNA analyses according to the quality check of the extracted RNA. Gene expression of 84 known cancer drug targets was evaluated by quantitative real-time PCR using 5 normal adrenal glands as reference. Protein expression was investigated for selected candidate drug targets by immunohistochemistry in 104 ACC samples, 11 adenomas and 6 normal adrenal glands. Efficacy of an available inhibitor of the most promising candidate was tested by functional in vitro experiments in two ACC cell lines (NCI-H295R and MUC1) alone or in combination with other drugs.
Most frequently overexpressed genes were TOP2A, IGF2, CDK1, CDK4, PLK4 and PLK1. Nuclear immunostaining of CDK1, CDK4 and PLK1 significantly correlated with the respective mRNA expression. CDK4 was chosen as the most promising candidate for functional validation as it is actionable by FDA-approved CDK4/6 inhibitors. ACC samples with copy number gains at CDK4 locus presented significantly higher CDK4 expression levels. The CDK4/6 inhibitor palbociclib showed a concentration- and time- dependent reduction of cell viability in vitro, which was more pronounced in NCI-H295R than in MUC1 cells. This was in line with higher CDK4 expression at western blot analysis in NCI-H295R cells. Furthermore, palbociclib was applied in combination with dual IGFR/IR inhibitor linsitinib showing a synergistic effect on reducing cell viability. In conclusion, this proof-of-principle study confirmed RNA profiling to be useful to discover potential drug targets. Detected drug targets are suitable to be investigated by immunohistochemistry in the clinical setting. Moreover, CDK4/6 inhibitors are promising candidates for treatment of a subset of patients with tumors presenting CDK4 copy number gains and/or overexpression, while linsitinib might be an interesting combination partner in patients with both IGF2 and IGF1R overexpression.
These results are intended as a basis for a validation study in a prospective cohort, further evaluation in vivo in suitable mouse models or testing in patients with ACC in clinical trials are needed and might improve the future management of patients with ACC in terms of precision medicine.
SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis.
NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs.
Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases.
Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses.