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The HIV protein, Vpr, is a multifunctional accessory protein critical for efficient viral infection of target CD4+ T cells and macrophages. Vpr is incorporated into virus particles and functions to transport the preintegration complex into the nucleus where the process of viral integration into the host genome is completed. This action is particularly important in macrophages, which as a result of their terminal differentiation and non-proliferative status, would be otherwise more refractory to HIV infection. Vpr has several other critical functions including activation of HIV-1 LTR transcription, cell-cycle arrest due to DCAF-1 binding, and both direct and indirect contributions to T-cell dysfunction. The interactions of Vpr with molecular pathways in the context of macrophages, on the other hand, support accumulation of a persistent reservoir of HIV infection in cells of the myeloid lineage. The role of Vpr in the virus life cycle, as well as its effects on immune cells, appears to play an important role in the immune pathogenesis of AIDS and the development of HIV induced end-organ disease. In view of the pivotal functions of Vpr in virus infection, replication, and persistence of infection, this protein represents an attractive target for therapeutic intervention. Numerous studies have reported that Vpr alters NF-kappa B signaling in various cells, however, the findings have so far been largely conflicting with reports both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells and address discrepancies that have been reported in the field. Our results show that Vpr expressed intracellularly is inhibitory to NF-kappa B, while extracelluar Vpr may have some stimulatory effects. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-alpha pathway, but not the LPS pathway, suggesting that multiple targets of Vpr may exist for NF-kappa B regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of I-kappa B-alpha in response to TNF-alpha stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Finally, our findings suggest that NF-kappa B regulation by Vpr is further changed by the presence of other HIV-1 components within the cells, as U1 cells lacking Vpr were unexpectedly less responsive to TNF-alpha than those cells that had normal Vpr expression levels. This data suggests that Vpr may serve an important role in vivo by selectively inhibiting immune activation while stimulating NF-kappa B mediated viral production in HIV-1 infected T-cells and myeloid cells. M-CSF is a cytokine that promotes monocyte differentiation and survival. When over-expressed, M-CSF contributes to pathology in a wide variety of diseases including osteoporosis, obesity, certain human cancers, and in HIV-1 infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1. In this study, our aim is to expand on the current knowledge of M-CSF regulation by NF-kappa B, a prominent transcription factor during inflammation and HIV-1 infection. Our results suggest that TNF-alpha promotes M-CSF secretion in macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-kappa B pathway, diminish this response. We identified four putative NF-kappa B and four C/EBP-beta binding sites within the M-CSF promoter. Our findings using M-CSF promoter constructs mutated at individual NF-kappa B locations suggest these sites are redundant with respect to M-CSF promoter regulation. TNF-alpha treatment promoted NF-kappa B p65 binding to the M-CSF promoter in PMA treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-kappa B response. In conclusion, our findings demonstrate that NF-kappa B induces M-CSF expression on a promoter level via multiple functional NF-kappa B binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages. The oncogenic potential of M-CSF receptor has been has been suggested over thirty years ago, however, few current studies have focused on the role of the receptor in AML. In a clinical trial for AML, Sunitinib was found to hold some efficacy for treating the disease. The authors hypothesized that the primary therapeutic target of Sunitinib in AML is FLT3 kinase. However, FLT3 inhibition alone has not been shown to recapitulate all the effects of Sunitinib in vitro and, furthermore, the drug is also known to have cross reactivity to other potential oncogenic receptors. In this study, we treated three myeloid cell lines, Mono-Mac 1, THP-1 and U937 with Sunitinib and a proprietary cFMS inhibitor from Johnson and Johnson to test the anti-cancer effect in of such treatment. We observed that only Mono-Mac 1 cells had diminished proliferation in vitro. Mono-Mac 1 cells had inhibited ERK as a result of cFMS inhibition and showed a dose dependent increase in cFMS expression with both Sunitinib and J&J cFMS-1 treatment. Our results suggest potential for cFMS as an important target of Sunitinib or other similar drugs AML, either independently or in combination with other targets. Alternatively, cFMS may be a marker for differentiation of AML and may be linked with responsiveness to certain therapeutics. In both cases, the future study of cFMS may produce more targeted therapeutic approaches and may be a suitable tool for the development of personalized medicine for AML. / Biomedical Neuroscience

Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, characterized by the accumulation of abnormally differentiated blast cells that is driven by leukemic stem cells (LSCs). In murine AML, Musashi-2 (MSI2), an RNA-binding protein and positive regulator of stemness, has been implicated in the propagation of disease. While its enhanced expression correlated with poor disease outcome for human AML patients, no study has yet examined its actual functional role in human leukemia. In normal human hematopoietic stem cells (HSCs), we have recently reported the inhibitory effects of MSI2 on the pro-differentiative aryl hydrocarbon receptor (AHR) signaling pathway as a mechanism for promoting self-renewal in HSCs. We hypothesized that elevated MSI2 is critical for maintenance of human AML and promotes unrestrained self-renewal of LSCs in part through constitutive repression of AHR signaling. Our work aimed to unravel the relationship between MSI2 and AHR in the human leukemic context and to determine if activation of AHR signaling can promote differentiation. Results confirmed that MSI2 is preferentially expressed in primary patient LSCs and is negatively correlated with the expression of AHR gene targets. Upon lentiviral knockdown of MSI2 in-vitro and in-vivo, leukemic growth was compromised and increased AHR signaling was observed. Circumventing the inhibitory role of MSI2 in AML, activation of AHR with a potent agonist impaired leukemic progenitor activity and proliferation. In-vivo studies employing reconstitution of immunodeficient mice with primary AML samples showed impairment of AML engraftment for a significant proportion of tested samples upon treatment with an AHR agonist. Overall, our findings from this project indicated that MSI2 is required for human AML propagation and that a decrease in MSI2 inhibitory effects on AHR signaling or direct activation of the AHR signaling pathway via a potent agonist can promote AML cell differentiation and loss. / Thesis / Master of Science (MSc) / The human blood system is sustained by a population of blood stem cells that are tightly regulated in their production of stem and differentiated cells. The Musashi-2 (MSI2) protein is a key regulator of blood stem cell identity through its inhibition of the aryl hydrocarbon receptor (AHR) signaling pathway. When there is dysregulation of blood cell homeostasis, blood malignancies such as acute myeloid leukemia (AML) may arise. In this work, the relationship between MSI2 and the AHR signaling pathway was explored within a myeloid leukemic context. It was shown that MSI2 imposes inhibitory effects on AHR to promote disease progression and that its reduction could help alleviate disease burden. Additionally, it was found that activation of the AHR signaling pathway could overcome the MSI2 differentiation block to create a therapeutic effect. Overall, the results of this project shed light on novel therapeutic strategies and targets for the treatment of AML.

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of age-related hematopoietic disorders characterized by ineffective and incomplete hematopoiesis leading to an increased risk of acute myeloid leukemia (AML). The development of accurate and easily used in vitro models is crucial for understanding the pathogenesis of MDS and identifying potential therapeutic targets. Induced pluripotent stem cells (iPSCs) can be used to study MDS due to their ability to differentiate into any cell type depending on the environment. The main limitation is that the blood progenitors produced by iPSCs are of a fetal state, which hinders modeling of MDS, a disease of older adulthood. This study aimed to optimize the maturation state of blood progenitors derived from iPSCs by induction of the micro-RNA let-7, which, we hypothesize will increase the maturation and adult phenotypic state of hematopoietic progenitors. iPSC lines were generated from healthy controls and samples containing the SRSF2 mutation, a common mutation in MDS, containing a doxycycline-inducible, stabilized let-7 transgene. A stepwise differentiation efficiently drove the iPSCs toward hematopoietic progenitors and, subsequently, other mature lineages. The hematopoietic progenitors were characterized by assessing the expression of specific cell surface markers and functional properties using flow cytometry, colony-forming assays, and multi-lineage differentiation abilities. These findings demonstrate the potential of using iPSC engineering to create a novel model for MDS and other age-biased disorders by inducing let-7 expression in iPSCs and, when differentiating them, exposing them to doxycycline to promote an adult cell phenotype. This approach offers a valuable potential tool for elucidating the molecular mechanisms underlying these disorders and exploring potential therapeutic interventions. / 2026-03-13T00:00:00Z

In acute myeloid leukemia (AML), between 15-20% of all patients harbor a somatic mutation in the isocitrate dehydrogenase 1 or 2 gene (IDH1 and IDH2). Therefore, these mutations are among the common ones in AML. However, the prognostic significance of mutated IDH in AML patients remains controversial. Several research groups reported distinct outcomes within specific patient subsets depending on the biological and clinical context. Additionally, the prognostic impact seems to be depended by the co-mutations, the specific location of the mutation (i.e., regarding the hotspot locations IDH1 R132, IDH2 R140, and IDH2 R172) as well as the applied treatment. Today, allogeneic hematopoietic stem cell transplantation (HSCT) remains the consolidation therapy with the highest chance of sustained remission for most younger and older AML patients. Even though many IDH mutated AML patients are consolidated by HSCT and several trials testing IDH inhibitors in a maintenance setting are active, very little data are available on the influence of IDH mutations at diagnosis and as measurable residual disease (MRD) marker in the HSCT context. The first aim of our study was to study the frequency of IDH mutations and assess their associations with other biological and pretreatment markers. In our cohort of 292 AML patients, who all received an HSCT for consolidation, we identified somatic IDH mutations in 70 (24%) patients. IDH1 mutations were found in 11.4% of the patients, all of which were R132 substitutions. Regarding mutations in the IDH2 gene, we identified 8.9% and 5.1% patients harboring a R140 or a R172 substitution, respectively. Generally, IDH mutated patients did not differ significantly from IDH wild type patients in our set regarding their biological characteristics with the exception that IDH mutated patients had significant higher bone marrow blasts at diagnosis. When we analyzed the mutational landscape of our cohort, we found that IDH mutated patients more frequently also harbored DNMT3A mutations, while RUNX1 mutations and TP53 mutations were found in lower frequencies. For the diagnostic bone marrow variant allele fractions (VAFs) associated with the three IDH mutation types, we observed a mutation specific pattern. While IDH2 R140 mutations clustered around a median VAF of about 50%, the VAFs for IDH1 R132 and IDH2 R172 mutations at diagnosis were significantly lower by median but showed a wider distribution. Next, we aimed to examine the prognostic value of different IDH1 and IDH2 mutations in AML patients that receive a consolidating allogeneic HSCT. Here, we observed no differences in the cumulative incidence of relapse (CIR), event-free survival (EFS), and overall survival (OS) according to the IDH mutational status at diagnosis. This also held up when we analyzed the three mutations, i.e., IDH1 R132, IDH2 R140, and IDH2 R172 mutations separately. Also in multivariable analyses, the diagnostic IDH mutation status did not significantly associate with outcomes after HSCT. However, we also analyzed the prognostic impact in the context of the ELN2017 classification and observed a distinct prognostic impact of the IDH mutations. With respect to outcome following HSCT consolidation in the ELN2017 Favorable-risk group, IDH mutation status did not influence outcomes. In the ELN2017 Intermediate-risk group, IDH mutated patients had an increased relapse rate compared to IDH wild-type patients. Nevertheless, this did not translate into significant shorter EFS or OS. Within the ELN2017 Adverse-risk group, IDH mutated patients had a lower CIR and by trend longer OS and EFS. The final major objective of our study was to analyze the role of IDH mutations as MRD marker in AML patients in CR prior to an allogeneic HSCT. In our cohort, 44 mutated patients had material for IDH mutation status detection on MRD level using digital droplet PCR (ddPCR) at HSCT available. DdPCr is a novel method that allows absolute quantification of gene mutations and/or expression without the necessity of standard curves. We established an assay that allows the quantification of IDH1 and IDH2 mutations with very high sensitivity and specificity. Of the 44 patients, 33 (75%) had detectable IDH MRD. The ddPCR based IDH mutation MRD positive patients were differently distributed (IDH1 R132: 65%; IDH2 R140: 94%; IDH2 R172: 44%). Interestingly, the VAFs at HSCT were much lower in IDH1 R132 (median 0.17%) and IDH2 R172 (median 0.20%) compared to IDH2 R140 (median 11.6%). Looking for association between IDH MRD status at HSCT and outcome, we observed a strong relapse association of IDH1 R132 positivity or IDH2 R172 positivity. Patients that were MRD positive for IDH1 R132 or IDH2 R172 mutations also had a shorter - though not significant - EFS and OS. Thus, clinically, the elimination of persisting IDH mutations – especially of IDH1 R132 and IDH2 R172 – before HSCT could be an important milestone towards a cure for these patients. On the other hand, IDH2 R140 MRD positivity at HSCT did not associate significantly with the CIR, EFS, and OS. Together with the previous mentioned finding of a higher VAF at diagnosis, we speculated that in our cohort, IDH2 R140 mutations behaved more like a clonal hematopoiesis-related aberrations.:Inhalt/Content Bibliographische Beschreibung/Bibliographic Description 1 Referat/Abstract 2 Einleitung/Introduction 3 Acute myeloid leukemia 3 Epidemiology 3 Clinical presentation and pathogenesis 3 Diagnostic workup and classification 3 Risk stratification and treatment 6 2022 European LeukemiaNet genetic risk classification 6 Standard Treatment 7 Measurable Residual Disease 10 Isocitrate Dehydrogenase 1 and 2 mutations 11 Definition 11 Pathology and Epidemiology 12 IDH mutations as prognostic markers 12 IDH inhibitors 13 Publikation/Publication 15 Zusammenfassung/Summary 22 Perspektive/Outlook 26 Literaturverzeichnis/References 27 Anlage/Supplemental Material 36 Referenz der Publikation/Reference of the Publication 55 Erklärung über den wissenschaftlichen Anteil des Promovenden 56 Erklärung über eigenständige Abfassung der Arbeit 58 Lebenslauf/Curriculum Vitae 59 Publikationen/Publications 61 Erst- und Letztautorschaften/First and Last authorship 60 Ko-Autorschaften/Co authorship 61 Reviews 66 Danksagung/Acknowledgments 67

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Hematopoiesis is a highly orchestrated process, in which a single hematopoietic stem cell (HSC) gives a rise to all blood cellular components. For myeloid and lymphoid development precise controlled expression of the PU.1 transcription factor is needed. Deletion of PU.1 gene in mouse is lethal and its dysregulation during hematopoietic differentiation is associated with blood malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS and AML are serious blood disorders characterized by expansion of immature blood cells and lack of differentiated functional cells. Not only genetic but also epigenetic aberrations represent a very important field for studying pathophysiology of leukemia genesis and dysregulation of the PU.1 gene represents intensively studied candidate mechanism. Modern therapy of selected MDS and subset of AML patients is based on treatment with DNA hypomethylating agent Azacytidine (AZA) interfering in PU.1 gene regulatory mechanism. However, poor response or resistance to this therapy often occurs. In this thesis we present data obtained from AZA-resistant clones of MDS/AML cell line OCI-M2. We analysed DNA methylation and DNA hydroxymethylation at the key regulatory element of the PU.1 gene (URE). We found that these epigenetic modifications at URE...

61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...

Allt fler banker tillämpar artificiell intelligens (AI) i syfte att bekämpa finansiell brottslighet. Med den ökade användningen av AI uppkommer etiska utmaningar som banker behöver hantera för att säkerställa en god etik vid nyttjande av AI vid finansiell brottsbekämpning. Syftet med studien var att undersöka vilka ställningstaganden som ligger till grund för hur banker som använder AI hanterar etiska utmaningar inom finansiell brottslighet. Studien bygger på en kvalitativ ansats med semistrukturerade intervjuer för insamling av empiri samt en litteraturstudie för att besvara frågeställningen. En tematisk analys har gjorts för hur banker hanterar etiska utmaningar vid nyttjandet av AI för att bekämpa finansiell brottslighet, vilket ledde till följande slutsatser: banker hanterar etik både på individuell och organisatorisk nivå genom att undvika partiskhet, följa lagkrav, vara transparenta gentemot kunder att de övervakas samt följa upp beslut fattade av AI. Studiens resultat diskuteras utifrån etiska förhållningssätt såsom utilitarism, pliktetik och dygdetik. / More and more banks are applying artificial intelligence (AI) to fight financial crime. With the increased use of AI, ethical challenges arise that banks need to handle in order to ensure good ethics when using AI when fighting financial crime. The purpose of the study was to investigate which stances are the basis for how banks that use AI handle ethical challenges in financial crime. The study is based on a qualitative approach with semi-structured interviews to gather empirical evidence and a literature study to answer the research question. A thematic analysis has been made of how banks deal with ethical challenges when using AI to fight financial crime, which led to the following conclusions: banks deal with ethics both at an individual and organizational level by avoiding bias, complying with legal requirements but using the exceptions that exist for combating money laundering, being transparent to customers that they are being monitored, following up on decisions made by AI. The study's results are discussed based on different ethical approaches such as utilitarianism, duty ethics and virtue ethics.

Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.