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Acute myeloid leukaemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify novel therapeutic targets in AML, I have optimized a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. This work led to the identification of 492 AML-specific cell-essential genes, including several established therapeutic targets such as that represent new clinically actionable candidates. I have validated selected genes using DOT1L, BCL2, MEN1 and many other genes genetic and pharmacological inhibition, and chose candidates for downstream studies. Both the epigenetic modifier KAT2A and SRPK1 as promising KAT2A and spliceosome kinase SRPK1 inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs while sparing normal hemopoietic stem-progenitor cells. My findings propose that KAT2A and SRPK1 inhibition should be investigated as new therapeutic strategies in AML and also provide a large number of novel genetic vulnerabilities of this leukaemia that can be pursued in downstream studies. As these screens were performed in immortalised AML cell lines, I then went on to develop a method for the performance of dropout screens in genotypically-defined primary murine AMLs developed in our lab and arising in Cas9-expressing mice. Through this work, I successfully carried out the first such screen in AML cells driven by mutant Npm1 (NPM1c) and Flt3-ITD, the commonest two-mutation combination in human AML. Downstream analysis of the results revealed the excellent potential of this type of screen and enabled me to investigate the molecular effects on mutant Npm1, which are currently poorly understood. Overall, my results demonstrate that unbiased CRISPR dropout screens can identify novel therapeutic targets in cancer while, in parallel, revealing novel biological insights.

Background. The majority of childhood acute myeloid leukemia (AML) patients lack a matched-related bone marrow transplant (BMT) donor in first remission. Procedure. Disease-free survival (DFS), overall survival (OS), relapse-free survival (RFS), and post-relapse outcome were evaluated for children with de novo AML on CCG 213 and the standard timing (ST) and intensive timing (IT) induction arms of CCG 2891 who were randomized to (intent-to-treat, ITT) or who received (as-treated, AT) only chemotherapy intensification. Results. Outcomes at 8 years post-induction in ITT analysis of chemotherapy intensification were as follows: 31% DFS, 43% OS on CCG 213; 34% DFS, 51% OS on CCG 2891 ST; 48% DFS, 56% OS on CCG 2891 IT. All toxic deaths during and following Capizzi II chemotherapy intensification on both protocols were in patients >3 years of age (P ≤ 0.001). Black race was a significant poor prognostic factor for OS (P = 0.008, hazard ratio: 1.74, 95% CI: 1.15-2.61). Overall 48% of patients on both trials relapsed and 19.1% of patients who relapsed on these trials survived. CR1 >12 months portends a much better OS for patients who relapse. Post-relapse treatment included BMT in 47% of patients. Conclusions. OS on CCG 2891 was superior to CCG 213 but equivalent between ST and IT arms due to better salvage rates post-relapse in ST patients. Overall survival for childhood AML in the absence of BMT in CR1 is influenced by duration of CR1 and by race.

Indiana University-Purdue University Indianapolis (IUPUI) / Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) cause constitutive activation of the receptor and confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that Shp2 interacts with FLT3-ITD via protein complexes at tyrosine (Y) 768, 955, and/or 969 and that Shp2 and PI3K work cooperatively to promote FLT3-ITD-induced leukemogenesis. Consistently, mutation of N51-FLT3 tyrosine 768 to phenylalanine reduced proliferation and levels of phospho-Erk compared to N51-FLT3-expressing cells while having no effect on levels of phospho-STAT5. In transplants, C3H/HeJ mice injected with either WT-FLT3-, N51-FLT3-, or N51-Y768F-expressing cells showed that mutation of Y768 had no effect on overall survival. In addition, pharmacologic inhibition of Shp2 with II-B08 or PI3K with GDC-0941 in N51-FLT3-expressing cells and primary patient samples showed decreased proliferation. A possible mechanistic explanation for reduced proliferation and selective reduction of P-Erk levels in the N51-FLT3-Y768-expressing cells is through decreased recruitment of Grb2, which participates with son of sevenless, SOS, to activate the RAS-Erk signaling pathway. The lack of improvement in overall survival could be due to preserved STAT5 signaling, as observed during in vitro experiments. Collectively, these data suggest that the tyrosine 768 residue plays an important role in phospho-Erk signaling in N51-FLT3-expressing cells, and that pharmacologic therapy with Shp2 or PI3K inhibitors may provide a novel treatment approach for FLT3-ITD positive AML. For future directions, we plan to treat mice with the Shp2 inhibitor, II-B08, the PI3K inhibitor, GDC-0941, or a combination to determine the effect on overall survival.

1 Atomic swap: a risk for AML regulation? Abstract This thesis deals with atomic swaps. Particularly it deals with their characteristics and relation to AML regulation. The aim of this thesis is to assess the risks that atomic swaps pose to AML regulation and the ways in which these risks can be addressed. The first part of this thesis briefly describes the concepts and technologies needed to understand atomic swaps. Among other things this part describes distributed databases, cryptographic keys, wallets, and virtual currency exchanges. This is followed by a description of the evolution of atomic swaps, how atomic swaps work and what are their advantages and disadvantages. The second part analyses the current regulation against money laundering and terrorist financing from the perspective of FATF, the European Union and the Czech Republic in relation to virtual currencies and atomic swaps. Particularly it focuses on the definition of virtual currencies and obliged entities. After the explanation how atomic swaps work and the analysis of AML regulation in relation to atomic swaps, it is possible to assess whether atomic swaps pose a risk to AML regulation. This is the focus of the third part of this thesis. It first introduces the issue of money laundering and the use of virtual currencies for illicit...

Acute myeloid leukemia (AML) is a highly fatal blood cancer that is characterized by disruption of healthy differentiation of stem cells into functional blood cells in the bone marrow. Most patients with AML consequently die from infections due to the lack of immune cells. For decades, the standard method of remission induction for AML has been chemotherapy using an antineoplastic drug known as AraC. However, even after successful remission induction, aggressive, refractory relapse occurs in the majority of patients within 3 years with dismal survival rates. Here, we sought to develop a genome-wide screening approach to determine the causative genes in AML relapse. In the developed procedure, protein-coding genes of the human genome are screened using a leading-edge technology known as CRISPR (clustered regularly interspaced short palindromic repeats) activation screening. This involves usage of a pooled guide RNA library that upregulates a unique gene for each individual AML cell. By treating these cells with AraC in a mouse xenograft model, the bone marrow will gradually be enriched with cells that carry a guide RNA for a relapse-conducive gene. By harvesting and sequencing all enriched guide RNAs at relapse, the causative genes in AML relapse can be determined. All parameters of the in vivo CRISPR-activation screen have been optimized, and the workflow from preparation to the end of screening has been detailed. Follow-up studies that will validate the results of the screen have also been described. The long-term goal of this developed screen is to elucidate the mechanisms of AML relapse and find ways to clinically target these pathways in conjunction with standard the AraC-based chemotherapy. / Thesis / Master of Science (MSc)

Despite advances in the understanding of cancer biology, most patients with acute myeloid leukemia (AML) still die of their disease. Improving risk-stratification and identifying new targets are important steps towards personalized medicine and outcome improvement. MicroRNAs, short non-coding RNAs that hybridize to their target messenger RNAs (mRNAs) and repress the expression of the encoded proteins, are known to be involved in physiological processes like cellular differentiation, proliferation and cell survival but also play an essential role in cancer, including AML. In this thesis we demonstrated that higher expression of a single microRNA miR-181a was associated with clinical outcome in cytogenetically normal AML (CN AML) patients. In multivariable models, higher expression of miR-181a was associated with achievement of complete remission (CR), with longer disease-free (DFS) and overall survival (OS) even in consideration of other validated prognostic clinical and molecular variables. Measurement of pretreatment levels of this microRNA may improve risk-stratification for AML patients. A genome-wide gene-expression signature gave biological insights into miR-181a associated AML, and provides a basis for further functional studies. Furthermore, as higher miR-181a expression associated with improved treatment response, increasing miR-181a levels by delivering synthetic miR-181a or by agents increasing endogenous levels of this microRNA in AML blasts may represent a novel and personalized therapeutic approach in AML.

High expression levels of the gene BAALC (brain and acute leukemia, cytoplasmic) are associated with poor prognosis in acute myeloid leukemia (AML) patients, but the underlying mechanisms are not yet understood. We evaluated the prognostic significance of expression levels of miR-3151, a newly discovered microRNA embedded in intron 1 of the BAALC gene, in a cohort of 179 older (≥60 years) cytogenetically normal AML (CN-AML) patients, in the context of established molecular markers and especially with regard to the possible interplay with its host gene BAALC. In multivariable analyses, high miR-3151 was associated with shorter disease-free and overall survival (OS), while higher BAALC expression strongly predicted failure of complete remission attainment and OS. Patients exhibiting both high miR-3151 and BAALC expression had worse outcome than patients expressing low levels of either one of the genes or both. Next, gene - and microRNA-expression profiles associated with miR-3151 expression were derived using microarrays, and a pathway analysis of the miR-3151 associated gene signature was performed using Ingenuity software. High miR-3151 expressers showed downregulation of genes involved in transcriptional regulation, post-translational modifications and cell-cycle control. Two genes of the ubiquitination pathway, FBXL20 and USP40, were experimentally validated as direct miR-3151 targets. In summary, we identified high expression levels of the intronic miR-3151 as a novel, independent prognosticator for poor outcome in CN-AML. Interestingly, miR-3151 impacted differently on outcome than its host gene BAALC; and the combination of both markers identified a patient subset with the poorest outcome, suggesting that the microRNA and its host gene contribute to clinical and prognostic features of CN-AML independently and through distinct mechanisms. This is the first example of the interplay of an intronic miR and its host gene in leukemia. Its discovery may have important biologic implications for future targeted treatment strategies.

Clonal diversity in cancer has been proposed as a mechanism underlying patient-to-patient variability in therapeutic response, as well as the variability in the likelihood and the time to relapse of acute myeloid leukemia (AML) and other cancers. As a neoplasm develops it often continues to mutate, diversifying into differing clonal populations. Darwinian evolutionary pressures such as inherent fitness imbalances, immune system interactions, and chemotherapy treatments target sensitive clones and drive competition between the clonal populations; selecting for dynamic and resistant cell lines. In this way clonal diversity is conceivable as an impediment to a complete remission with more populations offering more opportunities for therapy resistance. Bulk next generation sequencing (NGS) is currently used to assess clonal composition in leukemia but requires several broad assumptions be made, which can result in incorrect assessments of diversity. Factors such as differences in zygosity of mutations, convergent evolution, or contamination with wild-type/non-cancerous cells can artificially raise or lower reported variable allele frequencies (VAF), leading to errors in clonal assessments. To examine discrepancies between the actual clonal structure and the clonal structures determined through bulk sequencing we developed a novel method of sampling the cell population to identify concurrent mutations. We first created an in silico model which randomly draws cell samples from a simulated tumor multiple times and calculates the VAF for each mutant allele in each sample. By tracking the correlation of mutations between sample replicates, a clonal composition that is not observable from the bulk NGS VAF becomes apparent. We then created in vitro model tumors from AML cell lines, isolated low cell number samples via flow cytometry, and applied a multiplex/nested PCR protocol with pyrosequencing to quantify VAFs in each sample. Again, by calculating the correlation of mutant alleles between replicates, previously unseen with NGS characteristics of the clonal structure becomes evident. Population sampling analysis may potentially offer a solution for clarifying how we can interpret NGS clonal analyses.

Acute myeloid leukemia (AML) is a disease characterized by overproduction of abnormally differentiated, hyper-proliferative myeloid cells known as blasts in bone-marrow and blood. Our laboratory has previously demonstrated that loss of epigenetic repression by the polycomb repressive complex 2 (PRC2), which is mediated by complex member metal response element binding transcription factor 2 (MTF2), drives chemo-resistance resulting in refractory AML. In this study, to identify MTF2-specific synthetic lethal interactions, a genome-scale CRISPR Knock-out (GeCKO) synthetic lethal screen was performed in matched MTF2-deficient and rescued THP-1 cells both in the absence and presence of the induction chemotherapeutic cytarabine. Following careful analysis of screening data using specialized software, 104 highly significant MTF2-specific synthetic lethal interactions as well as 15 cytarabine-specific synthetic lethal interactions were identified. Reduced stringency upon analysis helped to identify an additional seven MTF2-specific synthetic lethal interactions that could be targeted with commercially available small-molecule inhibitors. Among eight small molecule inhibitors, two DNA Polymerase A/Ribonucleotide Reductase Catalytic Subunit M1 (POLA/RRM1) dual inhibitors (clofarabine and fludarabine) were shown to induce toxicity with specificity for MTF2-deficient THP-1 cells at low concentrations only in the absence of cytarabine. In the future, further testing of the therapeutic potential of clofarabine and fludarabine in treating MTF2-deficient AML will be conducted in patient derived bone-marrow aspirates which better represent the true clonal and hierarchical nature of this life-threatening malignancy. Furthermore, lentiviral delivery of short-hairpin RNAs (shRNAs) targeting highly significant, non-enzymatic MTF2 and cytarabine-specific synthetic lethal interactions will be performed in both THP-1 cells as well as in patient derived bone-marrow aspirates. Eventually, in vitro validated targets will be validated under in vivo conditions using a patient derived xenograft (PDX) preclinical animal model of AML using immunocompromised NOD scid gamma (NSG) mice.