Multifunctional Role of the PAX5-JAK2 Oncoprotein in B Cell Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common tumor in children and also occurs in adolescents and adults. Despite good prognosis in pediatric patient outcome, at least 15% of patients will suffer from relapse. Hence, a deep understanding of the molecular mechanisms underlying the onset and progression of this disease is needed to improve current therapeutic approaches. During the last decade, world-wide genome sequencing effort have identified mutations in many genes that play an important role in the development of B cell leukemia. One of these genes codes for the transcription factor PAX5 that is essential for the generation of the immune cell type referred to as B cell, whose normal task is to generate specific antibodies to protect us from acute infections. In one third of all B-ALL cases, one of the two PAX5 gene copies is lost, thus implicating PAX5 as a tumor suppressor in leukemia development. Another characteristic feature of B-ALL is the frequent occurrence of chromosomal translocations, which lead to gene fusions that code for novel chimeric transcription factors. A subset of B-ALL with poor prognosis is characterized by the PAX5-JAK2 translocation that generates a fusion transcription factor consisting of the DNA-binding domain of PAX5 linked to the catalytically active domain of the JAK2 kinase. JAK2 can activate gene expression by phosphorylating members of the STAT transcription factor family or the nuclear protein histone H3, thus resulting in the local generation of active chromatin. The aim of this project is to elucidate how the PAX5-JAK2 protein controls leukemia development and/or maintenance. For this purpose, we have recently generated a mouse model expressing the PAX5-JAK2 protein from the endogenous Pax5 gene. The PAX5-JAK2-expressing mice rapidly develop an aggressive B-ALL tumor, indicating that they mimic the role of PAX5-JAK2 in human leukemia. We will next investigate to what degree PAX5-JAK2 contributes to B-ALL development through activation of JAK-STAT signaling and/or activation of local chromatin at its target genes. We will identify and characterize PAX5-JAK2-regulated target genes by genome- wide gene expression, PAX5-JAK2 binding and chromatin profiling analyses of the mouse B- ALL tumor cells. CRISPR-Cas9-mediated mutagenesis will next be used to define the functional relevance of the top-ranked target genes for leukemia development. By analyzing the gene + expression profile of human PAX5-JAK2 B-ALLs, we will determine the degree to which the critical PAX5-JAK2 target genes identified in the mouse tumor model are also deregulated in the human disease. Collectively, these data will provide novel important insight into the molecular functions, by which the oncoprotein PAX5-JAK2 induces B cell leukemia, and may suggest improved therapeutic approaches for the treatment of this B-ALL tumor.

MULTIFUNCTIONAL ROLE OF THE PAX5-JAK2 ONCOPROTEIN IN B CELL LEUKEMIA B-cell acute lymphoblastic leukemia (B-ALL) is the most common tumor in children and also occurs in adolescents and adults. Despite good prognosis in pediatric patient outcome, at least 15% of patients will suffer from relapse. Hence, a deep understanding of the molecular mechanisms underlying the onset and progression of this disease is needed to improve current therapeutic approaches. During the last decade, world-wide genome sequencing effort have identified mutations in many genes that play an important role in the development of B cell leukemia. One of these genes codes for the transcription factor PAX5 that is essential for the generation of B cells, whose normal task is to generate specific antibodies to protect us from acute infections. In one third of all B-ALL cases, one of the two PAX5 gene copies is lost, thus implicating PAX5 as a suppressor of leukemia development. Another characteristic feature of B-ALL is the frequent occurrence of chromosomal translocations, which lead to gene fusions that code for novel chimeric transcription factors. A subset of B-ALL with poor prognosis is characterized by the PAX5-JAK2 translocation that generates a fusion transcription factor consisting of the DNA-binding domain of PAX5 linked to the catalytically active domain of the JAK2 kinase. The aim of this project was to elucidate how the PAX5-JAK2 protein controls leukemia development and maintenance. For this purpose, we have generated a mouse model expressing the PAX5-JAK2 protein from the endogenous PAX5 gene. The PAX5-JAK2-expressing mice rapidly developed an aggressive B-ALL tumor, indicating that they mimic the role of PAX5-JAK2 in human leukemia. Unexpectedly, all tumors expressed only the PAX5-JAK2 protein but not the full-length PAX5 protein, as the second, wild-type PAX5 gene was deleted in the tumor cells. Interestingly, the PAX5-JAK2 protein was only able to efficiently interact with its DNA binding sites in the absence of the full-length PAX5 protein. Hence, the strong selection pressure to lose the full-length PAX5 protein indicates that the PAX5-JAK2 protein fulfils an important oncogenic function in the nucleus of the tumor cell. As shown by mutation analysis, this oncogenic function depends on both the DNA-binding domain of PAX5 and the JAK2 kinase activity. Molecular analyses revealed that PAX5-JAK2 regulates gene expression indirectly by phosphorylating and thus activating the transcription factor STAT5 in the nucleus. Hence, PAX5-JAK2 is an important oncogenic driver of leukemia development by maintaining phosphorylated STAT5 levels in the nucleus. Importantly, leukemia formation in PAX5-JAK2-expressing mice was significantly delayed upon treatment with the clinically approved JAK2 kinase inhibitor Ruxolitinib. Our analysis of the PAX5-JAK2 mouse model therefore predicts that human patients with PAX5-JAK2+ leukemia may benefit from treatment with JAK2 kinase inhibitors.