Parallel genome-wide screens identify synthetic viable interactions between the BLM helicase complex and Fanconi anemia.

Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects

Exploratory Analysis of the Transcriptome of Myeloproliferative Neoplasms for Diagnostic and Therapeutic Applications

Myeloproliferative Neoplasien (MPN) sind eine seltene Form von Blutkrebsarten, die ihren Ursprung in defekten Stammzellen haben. Einige Besonderheiten sind ihre klonale Blutbildung (Hämatopoese) und eine Überproduktion von ausgereiften Blutzellen der myeloiden Entwicklungslinie. Die vorliegende Doktorarbeit befasst sich mit den BCR-ABL1 negativen MPN Krankheiten. Diese sind Essentielle Thrombozythämie (ET), Polycythämia vera (PV) und Primäre Myelofibrose (PMF). MPN Erkrankungen durchlaufen unterschiedliche Phasen. Meistens werden Patienten in einer chronischen Phase diagnostiziert, die sich durch einen stabilen Krankheitsverlauf definiert. In dieser Phase werden somatische Mutationen in den Genen JAK2, CALR und MPL in mehr als 90% von MPN Patienten gefunden. Diese sogenannten MPN spezifischen Mutationen tragen zu phänotypischen Ausprägungen der Krankheit bei. Patienten, die keine Mutation in diesen Genen aufweisen, werden als dreifach-negativ (‚triple-negative‘) bezeichnet. Die diagnostische Aufarbeitung dieser Patientengruppe wird durch diese Eigenschaft erschwert. Einige Patienten können von einem chronischen zu einem progressiven Krankheitsbild (‚accelerated phase‘) voranschreiten. Bei manchen Patienten kann zusätzlich eine sekundäre akute myeloide Leukämie AML (‚blast phase‘) auftreten. In beiden Fällen führt dies zu einem Anstieg an genetischer Komplexität und einer deutlich reduzierten Überlebenswahrscheinlichkeit. Die Mehrheit der therapeutischen Maßnahmen behandelt die Symptome der Patienten und befasst sich nicht mit der Ursache der Erkrankung, nämlich der defekten Stammzelle. In dieser Doktorarbeit wurde an einer Kohorte von 104 chronisch erkrankten MPN Patienten und 9 weiteren Patienten mit progressiver sekundärer AML ‚whole-transcriptome‘-Sequenzierung an Granulozyten RNA von Patienten durchgeführt. Verschiedene rechnergestützte Verfahrensschritte (‚workflows‘) wurden etabliert, um Klonalität in Granulozyten zu ermitteln, um Fusionsgene, Einzelvarianten, Insertionen und Deletionen in unseren Sequenzierdaten zu finden, um Defekte im Spleißen von Exonen zu detektieren und schließlich um potenzielle Neoantigene in unseren Daten zu identifizieren. Mit diesen workflows war es möglich einen neuen alternativen Weg zur Ermittlung von Klonalität in Krebszellen aller weiblichen MPN Patienten zu beschreiben. Insbesondere wird ein großer Nutzen für die Diagnose von dreifach-negativ Patienten erwartet. In chronisch sowie sekundär erkrankten AML Patienten wurde eine kleine Anzahl an nicht-rezidiven Fusionsgenen gefunden. Diese Doktorarbeit beschreibt eine komplexe Landschaft an Mutationen und eine hohe Häufigkeit an Mutationen im Gen SF3B1 in Patienten mit PMF. Eine Analyse von Defekten im Exon-Spleißen von Patienten mit SF3B1 Mutationen, definiert anomales 3‘Spleißen als den am häufigsten auftretenden Defekt. Viele dieser Defekte führen zu potenziellen Veränderungen in der Aminosäuresequenz eines Proteins. Alle bisher genannten Mutationen wurden in dieser Arbeit gesammelt, um schlussendlich Neoantigene zu identifizieren. Die Bestimmung von Neoantigenen beruht auf rechnergestützten Vorhersagen von Peptiden und MHC-I Molekülen, die spezifisch für jeden Patienten sind. Damit konnten Neoantigene in Patienten gefunden werden, die von CALR und MPL mutierten Proteinen stammen. Zusammengefasst konnten Neoantigene in 62% aller MPN Patienten ermittelt werden, Dieses Ergebnis stellt eine vielversprechende Ressource für personalisierte Immuntherapie für MPN Patienten dar.Myeloproliferative Neoplasms (MPNs) are stem cell derived hematological cancers characterized by a clonal hematopoiesis and an increase of differentiated blood cells of the myeloid lineage. The following thesis focuses on BCR-ABL1-negative MPNs, namely essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Several phases of disease evolution in MPN are recognized. Patients often present with a chronic phase defined by a stable disease course and appearance of somatic mutations. Disease driving mutations in JAK2, CALR, and MPL are found in the majority of ET, PV, and PMF diseases and shape the phenotypic landscape of MPN patients. Patients without mutations in MPN driver genes are referred to as ‘triple-negative and represent a group of patients with unmet need for concise diagnosis. Patients can progress from a chronic to an accelerated phase or to a blast phase (secondary acute myeloid leukemia sAML) defined by an increase in genetic complexity and worse survival. Most therapeutic interventions treat patient symptoms and do not eradicate the MPN disease cell. Whole transcriptome sequencing was performed on granulocytes RNA of 104 chronic MPN and 9 patients transformed to secondary AML. Several established and novel computational workflows were applied for determining clonality in granulocytes, calling and filtering fusion genes, single nucleotide variants (SNVs) and insertion and deletions (Indels), exploring aberrant splicing, and for discovery and systematic mining of neoantigens on our transcriptome dataset of MPN patients. The extent of granulocyte clonality was estimated in female MPN patients and thereby an alternative clonality assay was introduced, inclusive for all females and for the diagnostic benefit of triple-negative patients. The mutational landscape of the transcriptome of MPN patients was explored and characterized. A scarce number of non-recurrent fusions was found in chronic and secondary AML patients. MPN patients have a complex landscape of SNVs and indels with high frequency of SF3B1 mutations in PMF patients. Analysis of splicing defects identify aberrant 3 splicing as the most common splicing alteration in SF3B1 mutated PMF patients. A list of genes with putative protein altering defects caused by mis-spliced exons was described. Finally, all protein altering defects were collected and binding affinity was predicted for MHC class I molecules to 8, 9, and 10 mer peptides derived from altered proteins of different mutation classes. Putative neoantigens derived from CALR and MPL mutations were found. In general, 62% of MPN patients showed evidence of recurrent neoantigens providing a potential usage for targeted immunotherapy.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2019(VLID)366388

Myelomonocytic Skewing In Vitro Discriminates Subgroups of Patients with Myelofibrosis with A Different Phenotype, A Different Mutational Profile and Different Prognosis

Normal hematopoietic function is maintained by a well-controlled balance of myelomonocytic, megaerythroid and lymphoid progenitor cell populations which may be skewed during pathologic conditions. Using semisolid in vitro cultures supporting the growth of myelomonocytic (CFU-GM) and erythroid (BFU-E) colonies, we investigated skewed differentiation towards the myelomonocytic over erythroid commitment in 81 patients with myelofibrosis (MF). MF patients had significantly increased numbers of circulating CFU-GM and BFU-E. Myelomonocytic skewing as indicated by a CFU-GM/BFU-E ratio ≥ 1 was found in 26/81 (32%) MF patients as compared to 1/98 (1%) in normal individuals. Patients with myelomonocytic skewing as compared to patients without skewing had higher white blood cell and blast cell counts, more frequent leukoerythroblastic features, but lower hemoglobin levels and platelet counts. The presence of myelomonocytic skewing was associated with a higher frequency of additional mutations, particularly in genes of the epigenetic and/or splicing machinery, and a significantly shorter survival (46 vs. 138 mo, p < 0.001). The results of this study show that the in vitro detection of myelomonocytic skewing can discriminate subgroups of patients with MF with a different phenotype, a different mutational profile and a different prognosis. Our findings may be important for the understanding and management of MF

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia.

The role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy. Stefan N. Constantinescu and Vladimir Divoky are co-last Autho

CD22 CAR T-cell associated hematologic toxicities, endothelial activation and relationship to neurotoxicity

Background Hematologic toxicities, including coagulopathy, endothelial activation, and cytopenias, with CD19-targeted chimeric antigen receptor (CAR) T-cell therapies correlate with cytokine release syndrome (CRS) and neurotoxicity severity, but little is known about the extended toxicity profiles of CAR T-cells targeting alternative antigens. This report characterizes hematologic toxicities seen following CD22 CAR T-cells and their relationship to CRS and neurotoxicity.Methods We retrospectively characterized hematologic toxicities associated with CRS seen on a phase 1 study of anti-CD22 CAR T-cells for children and young adults with relapsed/refractory CD22+ hematologic malignancies. Additional analyses included correlation of hematologic toxicities with neurotoxicity and exploring effects of hemophagocytic lymphohistiocytosis-like toxicities (HLH) on bone marrow recovery and cytopenias. Coagulopathy was defined as evidence of bleeding or abnormal coagulation parameters. Hematologic toxicities were graded by Common Terminology Criteria for Adverse Events V.4.0.Results Across 53 patients receiving CD22 CAR T-cells who experienced CRS, 43 (81.1%) patients achieved complete remission. Eighteen (34.0%) patients experienced coagulopathy, of whom 16 had clinical manifestations of mild bleeding (typically mucosal bleeding) which generally subsided following CRS resolution. Three had manifestations of thrombotic microangiopathy. Patients with coagulopathy had higher peak ferritin, D-dimer, prothrombin time, international normalized ratio (INR), lactate dehydrogenase (LDH), tissue factor, prothrombin fragment F1+2 and soluble vascular cell adhesion molecule-1 (s-VCAM-1). Despite a relatively higher incidence of HLH-like toxicities and endothelial activation, overall neurotoxicity was generally less severe than reported with CD19 CAR T-cells, prompting additional analysis to explore CD22 expression in the central nervous system (CNS). Single-cell analysis revealed that in contrast to CD19 expression, CD22 is not on oligodendrocyte precursor cells or on neurovascular cells but is seen on mature oligodendrocytes. Lastly, among those attaining CR, grade 3–4 neutropenia and thrombocytopenia were seen in 65% of patients at D28.Conclusion With rising incidence of CD19 negative relapse, CD22 CAR T-cells are increasingly important for the treatment of B-cell malignancies. In characterizing hematologic toxicities on CD22 CAR T-cells, we demonstrate that despite endothelial activation, coagulopathy, and cytopenias, neurotoxicity was relatively mild and that CD22 and CD19 expression in the CNS differed, providing one potential hypothesis for divergent neurotoxicity profiles. Systematic characterization of on-target off-tumor toxicities of novel CAR T-cell constructs will be vital as new antigens are targeted.Trial registration number NCT02315612

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression