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In dieser Dissertation wird der MEK5/ERK5- Signalweg als möglicher Angriffspunkt in der zielgerichteten Melanomtherapie identifiziert. Die Adressierung von ERK5 bietet eine Alternative, um einer Resistenzentwicklung gegenüber Inhibitoren des MAPK- Signalwegs entgegenzuwirken. Das maligne Melanom ist ein hochaggressiver Tumor mit steigender Inzidenz. Zunehmende Sonnenstunden im Rahmen des Klimawandels mit erhöhter Belastung der Haut durch UV-Strahlung werden die Problematik des malignen Melanoms für den Menschen in den nächsten Jahren weiter zunehmen lassen.
Die Aktivierung des MEK5/ERK5- Signalwegs scheint eine Reaktion von Tumorzellen auf Therapiestress zu sein. Diese Aktivierung liefert den Melanomzellen einen Überlebensvorteil und verhindert ein langfristiges Therapieansprechen. ERK5 beeinflusst den Zellzyklus von Melanomzellen und ist somit möglicherweise von wichtiger Bedeutung in der Tumorgenese des malignen Melanoms.
Patienten mit NRAS- Mutation profitieren auffallend weniger von einer gezielten MEKi-Therapie als solche mit BRAF Mutation. Für ersteres Patientenkollektiv steht aktuell lediglich die Immuntherapie zur Verfügung, wodurch oft nur ein kurzes, progressionsfreies Intervall erreicht werden kann und die Patienten häufig unter schweren Nebenwirkungen leiden. Grund für die problematische Behandlung könnte das häufige Auftreten einer basalen ERK5- Aktivierung in NRAS- mutierten Melanomen sein. Diese Arbeit liefert eine positive Prognose über den Nutzen einer ERK5- Inhibition als Erweiterung des Therapieschemas. Diese These gilt auch für Melanompatienten mit einer BRAF- Mutation. Patienten, die an einem malignen Melanom erkrankt sind, weisen zu 80% eine Mutation in einem dieser beschriebenen Onkogene auf. Die Arbeit lässt darauf schließen, dass eine ERK5- Inhibition in der Therapie von beiden Gruppen erfolgreich sein könnte und somit das Leben nahezu aller Melanompatienten betrifft.
Despite accounting for only a small proportion of all skin cancers, malignant melanoma
displays a serious health risk with increasing incidence and high mortality rate. Fortunately,
advances in the treatment of malignant melanoma now prolong survival and enhance response
and treatment efficacy. Established biomarkers help evaluate disease progression and
facilitate choosing appropriate and individual treatment options. However, the need for easily
accessible and reliable biomarkers is rising to predict patient-specific clinical outcome.
Eosinophil infiltration into the tumor and high peripheral eosinophil counts prior and during
treatment have been associated with better response in patients for various cancer entities,
including melanoma. An analysis of a heterogeneous study cohort reported high serum ECP
levels in non-responders. Hence, eosinophil frequency and serum ECP as a soluble
eosinophil-secreted mediator were suggested as prognostic biomarkers in melanoma. We
examined whether melanoma patients treated with first-line targeted therapy could also benefit
from the effects of eosinophils. In total, 243 blood and serum samples from patients with
advanced melanoma were prospectively and retrospectively collected before and after drug
initiation. To link eosinophil function to improved clinical outcome, soluble serum markers and
peripheral blood counts were used for correlative studies using a homogeneous study cohort.
In addition, functional and phenotypical characterizations provided insights into the expression
profile and activity of freshly isolated eosinophils, including comparisons between patients and
healthy donors.
Our data showed a significant correlation between high pre-treatment blood eosinophil counts
and improved response to targeted therapy and by trend to combinatorial immunotherapy in
patients with metastatic melanoma. In accordance with previous studies our results links
eosinophil blood counts to better response in melanoma patients. High pre-treatment ECP
serum concentration correlated with response to immunotherapy but not to targeted therapy.
Eosinophils from healthy donors and patients showed functional and phenotypical similarities.
Functional assays revealed a strong cytotoxic potential of blood eosinophils towards
melanoma cells in vitro, inducing apoptosis and necrosis. In addition, in vitro cytotoxicity was
an active process of peripheral eosinophils and melanoma cells with bidirectional features and
required close cell-cell interaction. The extent of cytotoxicity was dose-dependent and showed
susceptibility to changes in physical factors like adherence. Importantly, we provide evidence
of an additive tumoricidal function of eosinophils and combinatorial targeted therapy in vitro. In
summary, we give valuable insights into the complex and treatment-dependent role of
eosinophils in melanoma. As a result, our data support the suggestion of eosinophils and their
secreted mediators as potential prognostic biomarkers. It will take additional studies to
examine the molecular mechanisms that underlie our findings.
Wilms tumor (WT) is the most common kidney cancer in childhood. It is a genetically heterogeneous tumor and several genetic alterations have been identified in WT patients. Recurrent mutations were found in the homeo-domain of SIX1 and SIX2 in high proliferative tumors (18.1% of the blastemal-type tumors) as well as in the microprocessor genes DROSHA and DGCR8 (18.2% of the blastemal-type tumors), indicating a critical role of the SIX-SALL pathway and aberrant miRNA processing in WT formation. Underlined by the fact that a significant overlap between mutations in DROSHA and SIX1 was found, indicating a synergistic effect.
To characterize the in vivo role of DROSHA and SIX mutations during kidney development and their oncogenic potential, I analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA or SIX1 carrying a tumor-specific E1147K or Q177R mutation, respectively.
The DROSHA mutation E1147K was predicted to act in a dominant negative manner. Six2-cre mediated deletion of Drosha in nephron progenitors led to a lethal phenotype with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-creERT2 resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth-retarded mice died within the first two months. This strong phenotype was consistent with the predicted dominant-negative effect of DROSHA-E1147K.
Analysis of the SIX1-Q177R mutation suggested that the mutation leads to a shift in DNA binding specificity instead of a complete loss of DNA binding. This may end up in subtle changes of the gene regulatory capacity of SIX1. Six2-cre mediated activation of SIX1-Q177R lead to a viable phenotype with no alterations or shortened life span. Yet a global activation of SIX1-Q177R mediated by Zp3-cre resulted in bilateral hydronephrosis and juvenile death of the mice.
To mimic the synergistic effect of DROSHA and SIX1 mutations, I generated compound mutants in two combinations: A homozygous deletion of Drosha combined with an activation of SIX1-Q177R and a compound mutant with activation of DROSHA-E1147K and SIX1-Q177R. Each mouse model variant displayed new phenotypical alterations. Mice with Six2-cre mediated homozygous deletion of Drosha and activation of SIX1-Q177R were not viable, yet heterozygous deletion of Drosha and activation of SIX1-Q177R led to hydronephrosis, proteinuria and an early death around stage P28. Combined activation of DROSHA-E1147K and SIX1-Q177R under Six2-cre resulted in proteinuria, glomerulosclerosis and lesions inside the kidney. These mice also suffered from juvenile death. Both mouse models could confirm the predicted synergistic effect.
While these results underscore the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.
Despite the advancement in the treatment from genotoxic drugs to more targeted therapies, multiple myeloma (MM) remains incurable. MM is known for its complex genetic heterogeneity as different genetic lesion accrue over the course of the disease. The current work focuses on the functional analysis of genetic lesions found at the time of diagnosis and relapse and their potential role regarding therapy response and refractory disease. Genetic lesions involving tumor suppressor gene TP53, are found at diagnosis and tend to accrue during disease progression. Different types of mono- and biallelic TP53 alterations were emulated in the AMO1 cell line model, were functionally characterized and tested for their potential role in therapy response. Both types of single hit TP53 alteration (deletion 17p and TP53 point mutations) were found to have similar adverse effects on the functionality of the p53 system and response to genotoxic drugs which were completely abolished in the case of double hit TP53 alterations (no p53 expression, or mutant overexpression in wild type TP53 deletion background). Whereas, sensitivity to proteasome inhibitors remained unaltered. Using the clonal competition assay (CCA), single TP53 hit clones were found to have a fitness advantage over wildtype cells. Proliferative cell fitness was further enhanced in double hit TP53 clones, as they dominated wildtype and single hit TP53 clones in the CCA. Presence of external selection pressure in the form of low dose melphalan expedited the intrinsic fitness advantage. Alterations found in CUL4B, a component of CRL4-CRBN protein complex, a target of immunomodulatory drugs (IMiDs), were also functionally analyzed in the current study. Hotspot mutations and mutations found in IMiDs refractory patients were modelized in L363 cells and their role in IMiDs sensitivity was studied. CUL4B mutations were found not to be involved in providing lenalidomide resistance to the cell, whereas knocking CUL4B out was observed to provide negative fitness to the cells in CCA. In the presence of external selection pressure, these clones showed fitness, which was lost in the case of lenalidomide withdrawal. This shows that some alterations may play a role in refractory patients only in the presence of therapy, and as soon as therapy is discontinued, these altered clones may disappear such as clones with alterations in CUL4B. On the other hand, some alterations provide drug-independent intrinsic positive fitness, however, be further enhanced by drug exposure, such as seen in case of TP53 altered clones. Therefore, close monitoring and functional analysis of evolving clones is desired during disease progression, as it can be helpful in therapeutic guidance to achieve a better outcome for patients.
These days, treatment of melanoma patients relies on targeted therapy with BRAF/MEK inhibitors and on immunotherapy. About half of all patients initially respond to existing therapies. Nevertheless, the identification of alternative therapies for melanoma patients with intrinsic or acquired resistance is of great importance. In melanoma, antioxidants play an essential role in the maintenance of the redox homeostasis. Therefore, disruption of the redox homeostasis is regarded as highly therapeutically relevant and is the focus of the present work.
An adequate supply of cysteine is essential for the production of the most important intracellular antioxidants, such as glutathione. In the present work, it was investigated whether the depletion of cysteine and glutathione is therapeutically useful. Depletion of glutathione in melanoma cells could be achieved by blocking cysteine supply, glutathione synthesis, and NADPH regeneration. As expected, this led to an increased level of reactive oxygen species (ROS). Surprisingly, however, these changes did not impair the proliferation and survival of the melanoma cells. In contrast, glutathione depletion led to cellular reprogramming which was characterized by the induction of mesenchymal genes and the repression of differentiation markers (phenotypic switch). This was accompanied by an increased migration and invasion potential which was favored by the induction of the transcription factor FOSL1. To study in vivo reprogramming, Gclc, the first and rate-limiting enzyme in glutathione synthesis, was knocked out by CRISPR/Cas9 in murine melanoma cells. The cells were devoid of glutathione, but were fully viable and showed a phenotypic switch, the latter only in MITF-expressing B16F1 cells and not in MITF-deficient D4M3A.781 cells. Following subcutaneous injection into immunocompetent C57BL/6 mice, Gclc knockout B16F1 cells grew more aggressively and resulted in an earlier tumor onset than B16F1 control cells.
In summary, this work demonstrates that inhibition of cysteine supply and thus, glutathione synthesis leads to cellular reprogramming in melanoma. In this context, melanoma cells show metastatic capabilities, promoting a more aggressive form of the disease.
The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009).
This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo.
In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.
Machine-Learning-Based Identification of Tumor Entities, Tumor Subgroups, and Therapy Options
(2023)
Molecular genetic analyses, such as mutation analyses, are becoming increasingly important in the tumor field, especially in the context of therapy stratification. The identification of the underlying tumor entity is crucial, but can sometimes be difficult, for example in the case of metastases or the so-called Cancer of Unknown Primary (CUP) syndrome. In recent years, methylome and transcriptome utilizing machine learning (ML) approaches have been developed to enable fast and reliable tumor and tumor subtype identification. However, so far only methylome analysis have become widely used in routine diagnostics.
The present work addresses the utility of publicly available RNA-sequencing data to determine the underlying tumor entity, possible subgroups, and potential therapy options. Identification of these by ML - in particular random forest (RF) models - was the first task. The results with test accuracies of up to 99% provided new, previously unknown insights into the trained models and the corresponding entity prediction. Reducing the input data to the top 100 mRNA transcripts resulted in a minimal loss of prediction quality and could potentially enable application in clinical or real-world settings.
By introducing the ratios of these top 100 genes to each other as a new database for RF models, a novel method was developed enabling the use of trained RF models on data from other sources.
Further analysis of the transcriptomic differences of metastatic samples by visual clustering showed that there were no differences specific for the site of metastasis. Similarly, no distinct clusters were detectable when investigating primary tumors and metastases of cutaneous skin melanoma (SKCM).
Subsequently, more than half of the validation datasets had a prediction accuracy of at least 80%, with many datasets even achieving a prediction accuracy of – or close to – 100%.
To investigate the applicability of the used methods for subgroup identification, the TCGA-KIPAN dataset, consisting of the three major kidney cancer subgroups, was used. The results revealed a new, previously unknown subgroup consisting of all histopathological groups with clinically relevant characteristics, such as significantly different survival. Based on significant differences in gene expression, potential therapeutic options of the identified subgroup could be proposed.
Concludingly, in exploring the potential applicability of RNA-sequencing data as a basis for therapy prediction, it was shown that this type of data is suitable to predict entities as well as subgroups with high accuracy. Clinical relevance was also demonstrated for a novel subgroup in renal cell carcinoma. The reduction of the number of genes required for entity prediction to 100 genes, enables panel sequencing and thus demonstrates potential applicability in a real-life setting.
Identifying novel driver genes in cancer remains a crucial step towards development of new therapeutic approaches and the basic understanding of the disease.
This work describes the impact of the AP1 transcription activator component FOSL1 on melanoma maintenance. FOSL1 is strongly upregulated during the progression of melanoma and the protein abundance is highest in metastases. I found that the regulation of FOSL1 is strongly dependent on ERK1/2- and PI3K- signaling, two pathways frequently activated in melanoma. Moreover, the involvement of p53 in FOSL1 regulation in melanoma was investigated. Elevated levels of the tumor suppressor led to decreased FOSL1 protein levels in a miR34a/miR34c- dependent manner.
The benefit of elevated FOSL1 amounts in human melanoma cell lines was analyzed by overexpression of FOSL1 in cell lines with low endogenous FOSL1 levels. Enhanced levels of FOSL1 had several pro-tumorigenic effects in human melanoma cell lines. Besides increased proliferation and migration rates, FOSL1 overexpression induced the colony forming ability of the cells. Additionally, FOSL1 was necessary for anchorage independent growth in 3D cell cultures. Microarray analyses revealed novel downstream effectors of FOSL1. On the one hand, FOSL1 was able to induce the transcription of different neuron-related genes, such as NEFL, NRP1 and TUBB3. On the other hand, FOSL1 influenced the transcription of DCT, a melanocyte specific gene, in dependence of the differentiation of the melanoma cell line, indicating dedifferentiation.
Furthermore, FOSL1 induced the transcription of HMGA1, a chromatin remodeling protein with reprogramming ability, which is characteristic for stem cells. Consequently, the influence of HMGA1 on melanoma maintenance was investigated. In addition to decreased proliferation and reduced anoikis resistance, HMGA1 knockdown reduced melanoma cell survival. Interestingly, the FOSL1 induced pro-tumorigenic effects were demonstrated to be dependent on the HMGA1 level. HMGA1 manipulation reversed FOSL1 induced proliferation and colony forming ability, as well as the anchorage independent growth effect.
In conclusion, I could show that additional FOSL1 confers a clear growth benefit to melanoma cells. This benefit is attributed to the induction of stem cell determinants, but can be blocked by the inhibition of the ERK1/2 or PI3K signaling pathways.
The transcription factor NRF2 is considered as the master regulator of cytoprotective and ROS-detoxifying gene expression. Due to their vulnerability to accumulating reactive oxygen species, melanomas are dependent on an efficient oxidative stress response, but to what extent melanomas rely on NRF2 is only scarcely investigated so far. In tumor entities harboring activating mutations of NRF2, such as lung adenocarcinoma, NRF2 activation is closely connected to therapy resistance. In melanoma, activating mutations are rare and triggers and effectors of NRF2 are less well characterized.
This work revealed that NRF2 is activated by oncogenic signaling, cytokines and pro-oxidant triggers, released cell-autonomously or by the tumor microenvironment. Moreover, silencing of NRF2 significantly reduced melanoma cell proliferation and repressed well-known NRF2 target genes, indicating basal transcriptional activity of NRF2 in melanoma. Transcriptomic analysis showed a large set of deregulated gene sets, besides the well-known antioxidant effectors. NRF2 suppressed the activity of MITF, a marker for the melanocyte lineage, and induced expression of epidermal growth factor receptor (EGFR), thereby stabilizing the dedifferentiated melanoma phenotype and limiting pigmentation markers and melanoma-associated antigens. In general, the dedifferentiated melanoma phenotype is associated with a reduced tumor immunogenicity. Furthermore, stress-inducible cyclooxygenase 2 (COX2) expression, a crucial immune-modulating gene, was regulated by NRF2 in an ATF4-dependent manner. Only in presence of both transcription factors was COX2 robustly induced by H2O2 or TNFα. COX2 catalyzes the first step of the prostaglandin E2 (PGE2) synthesis, which was described to be associated with tumor immune evasion and reduction of the innate immune response.
In accordance with these potentially immune-suppressive features, immunocompetent mice injected with NRF2 knockout melanoma cells had a strikingly longer tumor-free survival compared to NRF2-proficient cells. In line with the in vitro data, NRF2-deficient tumors showed suppression of COX2 and induction of MITF. Furthermore, transcriptomic analyses of available tumors revealed a strong induction of genes belonging to the innate immune response, such as RSAD2 and IFIH1. The expression of these genes strongly correlated with immune evasion parameters in human melanoma datasets and NRF2 activation or PGE2 supplementation limited the innate immune response in vitro.
In summary, the stress dependent NRF2 activation stabilizes the dedifferentiated melanoma phenotype and facilitates the synthesis of PGE2. As a result, NRF2 reduces gene expression of the innate immune response and promotes the generation of an immune-cold tumor microenvironment. Therefore, NRF2 not only elevated the ROS resilience, but also strongly contributed to tumor growth, maintenance, and immune control in cutaneous melanoma.
Die Therapiemöglichkeiten für Patienten im Melanom Stadium IV sind nach wie vor begrenzt und die Erkrankung nur selten heilbar. Eine mögliche Ziel¬struktur in der Melanom¬therapie der Zukunft könnte das im Melanom häufig genetisch wild¬typisch vorliegende p53 sein.
Für vorliegende Arbeit wurden humane Melanomzelllinien, welche stabil mit einem p53-Reportergenkonstrukt transduziert waren, hinsichtlich ihrer p53-Expression, -Akti-vität und -Akti¬vierbarkeit untersucht. Alle verwendeten Melanom¬zell¬¬¬linien exprimierten p53 un¬ab¬hängig vom p53-Mutations¬status. Drei der sieben untersuchten p53-wild¬typischen Melanomzelllinien zeigten keine oder nur sehr niedrige p53-Reporter¬gen¬aktivität. Die anderen vier p53-wildtypischen Zelllinien dagegen waren durch hohe, mittels p53-Knockdown unterdrückbare Reportergen¬expression gekennzeichnet. Die Proliferation dieser Zellen in Gegenwart von aktivem p53 belegt, dass Melanomzellen eine hohe Toleranz gegenüber diesem Tumor¬suppressor besitzen können. Eine weitere Steige¬rung der p53-Expression und -Aktivität durch die Hemmung von MDM2 (mouse double minute 2) mit der Substanz Nutlin-3a führte in den Zellen mit messbarer p53-Aktivität jedoch zu einem G1-Zell¬zyklusarrest. Dies belegt die prinzipielle Eignung von p53 als mögliche thera¬peutische Zielstruktur. Aufgrund ihrer schlechten Biover¬füg¬barkeit und hohen Toxizität gelten MDM2-Inhibitoren bisher aber als ungeeignet für den klinischen Einsatz.
Die Reduktion hoher therapeutischer Nebenwirkungen könnte durch eine Melanom-spezifische Reaktivierung von p53 gelingen. Eine mögliche negativ-modulierende Wirkung des Mela¬¬nom¬¬markers TRP2 (tyrosinase-related-protein 2) auf p53 wurde im Jahr 2010 von Sendoel et al. nach Unter¬suchungen am Fadenwurm C. elegans vorgeschlagen. TRP2 wird beim metastasierten Melanom in mehr als 80 % der Fälle exprimiert und wäre, handelte es sich beim Melanom um einen weit verbreiteten Regulationsmechanismus, ein interessantes Zielprotein, um die Aktivität von p53 zu steigern. Die dargestellten Ergeb¬nisse zeigen, dass TRP2 zwar in vier von fünf Melanomzelllinien exprimiert wurde, die Unterdrückung der TRP2-Expression jedoch weder spezifischen Einfluss auf die p53-Expression noch auf die p53-Reportergenaktivität zeigte. Auch das veränderte Wachs¬tums¬¬¬verhalten der Zellen nach Unterdrückung von TRP2 mittels drei unterschiedlicher shRNAs konnte im Rescue-Experiment, bei dem TRP nach seinem Knockdown ektop exprimiert wurde, keinem spezifischen Effekt von TRP2 auf die p53-Expression oder p53-Reporteraktivität zugeordnet werden. Auch in der TRP2-negativen Zelllinie führte die ektope TRP2-Expression nicht zu einer verminderten p53-Expression oder -Aktivität. Für das im Gegensatz zu MDM2 deutlich melanomspezifischere TRP2 konnte demensprechend kein sicherer regulatorischer Zusammenhang mit p53 dargestellt werden.
Weitere Untersuchungen müssen zeigen, welche Bedeutung wildtypischem p53 im Melanom zukommt und ob sich weitere mögliche p53-Regulatoren als therapeutische Angriffspunkte eignen.