Browsing by Subject "DDX3X"

Burkitt lymphoma (BL) is an aggressive germinal center (GC) B cell derived lymphoma. BL accounts for 40% of pediatric lymphoma cases in the United States and over half of all pediatric malignancies in Sub Saharan Africa. BL is characterized by the t(8;14) chromosomal translocation that results in MYC overexpression. Translocation of MYC alone is insufficient to induce lymphomagenesis; additional genetic mutations are required. A better functional characterization of the genetic drivers of BL will lend insight into pathways that drive Burkitt lymphomagenesis, providing an opportunity to identify novel drug targets and develop improved therapies.

In order to identify novel genetic drivers of BL our lab previously sequenced 101 BL tumors with paired normal samples. We found that DDX3X is recurrently mutated in 46% of BL tumors, making it the third most commonly mutated gene in BL. We observed that DDX3X mutations are either truncating mutations (22%) or missense mutations (88%) that cluster around the two highly conserved functional domains of the protein. Based on the non-focal distribution of missense mutations throughout the DDX3X coding sequence and the high presence of truncation mutations in BL tumors, we hypothesize that in GC B cells DDX3X acts as a tumor suppressor gene whose normal function is destroyed by BL associated mutations, facilitating lymphomagenesis.

While DDX3X is frequently mutated in many types of cancer, its role in malignancy is poorly understood. In this study we focused on elucidation of the role of DDX3X in the specific context of GC B cells from which BLs arise. To test the hypothesis that DDX3X normally functions as a tumor suppressor we modeled DDX3X deficiency in normal and malignant GC B cells using parallel in vitro and in vivo approaches. First, we created transgenic Ddx3x deficiency mouse models with and without MYC overexpression. This approach allowed us to study DDX3X in a system that models the complexities of the immune system on a genetically defined background. Second, we used genome editing to precisely delete DDX3X expression in BL cell lines. BL is a genetically complex disease and the the use of BL cell lines provides allowed us to study the role of DDX3X on a genetic background typical of BL tumors. We then characterized both DDX3X loss of function models with respect to cellular processes related to tumor development. Third, we performed cross-linking immunoprecipitation with sequencing (CLIP-Seq) to identify the RNA targets of DDX3X in the germinal center.

Our study lead to unexpected results regarding the contribution of DDX3X to Burkitt lymphomagenesis, and highlighted an important role for DDX3X in B cell development. We found that Ddx3x deficiency in a mouse model of BL increased the time to tumor development by reducing the global B cell population available for malignant transformation. In tandem we found that Ddx3x deficiency at the GC B cell stage significantly reduced the GC B cell population in multiple lymphoid tissues, regardless of MYC status. Interestingly, we found that Ddx3x deficiency in pre-B cells expanded the pre-B cell population but decreased the population of later B cell stages. We then confirmed that the reduction of GC B cells in response to Ddx3x deficiency was not due to defects in GC B cell migration, germinal center architecture, cell cycle progression, or apoptosis. These combined data suggest an essential function for DDX3X in B cell development, particularly at the pre-B cell and GC B cell stages.

Similar to in the mice, in cell culture we also found that DDX3X loss did not significantly alter apoptosis or cell cycle progression. We found some evidence that DDX3X may play a role in DNA damage repair but these results were not consistent across conditions. Lastly, we identified DDX3X targeted RNA binding partners using CLIP-Seq. Our data corroborates previously published CLIP-Seq experiments showing that DDX3X binds numerous RNAs involved in translation and RNA processing. Additionally, we identified for the first time that DDX3X binds RNAs falling in the BRCA1 and ATM gene sets. Further experimentation is needed to determine the role DDX3X plays in these pathways with relationship to Burkitt lymphomagenesis.

The cerebral cortex is an anatomically complex brain structure that controls our higher cognitive functions such as abstract thought and language. The cortex is largely shaped during embryonic development when radial glial progenitors divide and differentiate to produce neurons. Neurons are organized into 6-layers through migration guided by the structural support of radial glial cells. Disruptions in progenitor proliferation or neuronal migration underly diverse neurodevelopmental disorders with life-long impacts on cognitive, psychiatric, and motor functions. Developmental mechanisms that build the brain are regulated by precise gene-expression networks. Here, we uncover two novel layers of post-transcriptional mRNA regulation in the developing cortex. First, we used model models to describe a widespread phenomenon of mRNA localization to the distal structures—the basal process and endfeet—of radial glial progenitors. With live imaging approaches, we detected active mRNA transport to and local translation within radial glial endfeet. Transcriptomic and proteomic analyses revealed that endfeet contain cytoskeletal, signaling, and proteostasis factors that may be locally and dynamically controlled. The second line of investigation focused on the RNA-helicase DDX3X which is frequently mutated in neurodevelopmental disorders. We discovered that Ddx3x is required both in progenitor differentiation and neuronal migration in the developing mouse cortex. DDX3X mutations varied from loss-of-function to missense alleles, and a subset of missense variants caused severe cortical malformations in patients. Biochemical and cell biological assays revealed that dominant missense mutations reduced DDX3X helicase activity and induced formation of RNA-protein aggregates associated with impaired translation, uncovering novel pathologies underlying developmental disorders. Together, these studies extend our understanding of post-transcriptional regulation in brain development and neurodevelopmental disorders.