Gene regulation in normal and malignant B-lineage cells

Abstract: B-cells are the central players of humoral immunity. It is known that their development is orchestrated by a small group of transcription factors including the E-proteins and the FOXO proteins, yet this process is far from completely understood. Similarly, our understanding of the gene regulatory networks in transformed malignant B-lineage cells including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) tumor cells remains incomplete. As a result of the development of next generation sequencing (NGS) and bioinformatic tools, delving into these biological questions on a genome-wide level has become an attainable goal. It is now possible to perform whole genome sequencing, transcriptional profiling, and other assays at a lower price and higher depth than ever before. Here, we have used different NGS and bioinformatic techniques, in conjunction with molecular assays to gain a further understanding into the regulation of B-lineage cells in health and disease. In the first study we utilized conditional knockout mice to study the role of the E-proteins E2- 2 and HEB in the hematopoietic system. Characterizing the hematopoietic system using FACS, RNAseq, and ChIPseq, we found that the combined loss of E2-2 and HEB mainly affected the B-, T-, and pDC-lineages. We concluded that E2-2 and HEB are indispensable for humoral immunity while not playing a major role for the development of the other blood lineages. In study II we made use of conditional knockout FoxO1 and FoxO3 mice to study the impact of the combined loss of FOXO1 and FOXO3. We performed RNAseq, ATACseq, ChIPseq, and phenotypic characterization by FACS. Our results showed that FOXO1 and FOXO3 are essential for early B-cell development, where they are critical for enforcing the early B-cell gene regulatory program on a mainly pre-established chromatin landscape. In studies III and IV we investigated the very early and late effects of ibrutinib on CLL patients by performing RNAseq and analysis on plasma protein levels. Our results demonstrated that some of the changes caused by ibrutinib happened at the latest within nine hours of administration. These changes were not all orchestrated by the CLL cells, as not all plasma proteins identified were expressed by the tumor cells. We also found that some of the biomarkers increasing after treatment were associated with cardiovascular disease and potentially could be involved in causing atrial fibrillation in ibrutinib-treated patients. Overall, ibrutinib rapidly affects transcription and plasma protein levels and its effects have a long-term impact. In study V we analyzed FACS sorted MM cells using linked-read whole-genome sequencing (lrWGS). In comparison to FISH based genetics performed in clinical routine, we were able to find 94% of known translocations and 96% of CNVs. Furthermore, we also detected >150 additional SVs and CNVs, some of which are known to be associated with prognosis. Overall, we demonstrated that good quality data can be obtained with this method and that both private and recurrent events can be identified. In conclusion, in these studies we have gained further understanding on the E-proteins, FOXO factors, the effect of ibrutinib in CLL patients, and how lrWGS can be used to genetically characterize patients with hematological malignancies

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