Sequencing-based examination of childhood leukemogenesis [electronic resource] : from circular RNA to single-cell genomic dissection

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and although outcomes have markedly improved over the past 50 years, it remains a leading cause of pediatric cancer-related morbidity and mortality. Studies have catalogued many of the recurrent genetic alterations that occur during the development of childhood ALL, but those investigations have not identified a definitive underlying etiology. Our current understanding is that childhood leukemogenesis begins with genetic changes in utero that interact with germline genetic predisposition to progress to ALL in a fraction of patients during childhood. Although secondary events that result in disease progression have not been identified, late exposure to common infections is a strong risk factor. To acquire a better understanding of childhood leukemogenesis, we leveraged the throughput of next-generation sequencing to globally evaluate the nucleic acid content of leukemia cells. Our initial study aimed to identify genomic structural rearrangements, as well as viral genomes or transcripts in ALL samples using RNA-seq. Through the development of novel experimental and computational methods, we surprisingly identified circular RNA isoforms are much more abundant than previously appreciated. Further work showed that they are present in normal human cells, other model organisms, and human plasma. Characterization of circular splicing of ASXL1, a gene commonly mutated in leukemia that contains a conserved circular isoform, found that the transcript is spliced into its circular isoform much less efficiently than its linear counterpart. However, we identified intronic repeat sequences, as well as exonic regions that iv are required for efficient circular splicing suggesting these molecules, which are estimated to comprise 1% of the human transcriptome, may have evolved distinct biological functions. We next used the single-allele readout of next-generation sequencing, as well as the automation of microfluidic devices to probe the genetic diversity of leukemia samples. We first discovered continued ongoing changes in the immunoglobulin locus of ALL cells using immune repertoire sequencing, which has helped improve ALL disease monitoring strategies. We then physically isolated and amplified the genomes of individual cells using microfluidics to identify mutation co-segregation patterns, as well as the clonal structures of ALL samples. With the experimental and clone-deciphering algorithms developed, we were able to determine that co-dominant clones are present in most patients, KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and there are clone-specific punctuated cytosine mutagenesis events. The latter finding may lead back to the identification of a viral etiology, as the mutations have signature of APOBEC, a cytosine deaminase that is induced to provide innate anti-viral defense. Taken together, these studies have provided new paths to better understand general questions about posttranscriptional regulation, as well as the specific events that result in the development of childhood leukemias. Our deep characterization of linear and circular splicing has raised fundamental questions about the diversity of alternative splicing, as well as the importance of the resulting processed RNA in posttrancriptional regulation. Our high resolution dissection of clonal heterogeneity in childhood ALL has led to questions that could clarify the underlying etiology and pathogenesis, including the roles for APOBEC proteins, clonal symbiosis and competition, and the evolution of clonal structures that occurs after changes in selection pressure. It will take hard work, creativity, and continued technology development to decipher definitive roles of v posttranscriptional regulation and genomic heterogeneity in normal and malignant cellular biology.

Advisor: Brown Patrick O'Reilly
Sarnow P. (Peter) primary advisor. Thesis advisor
Chang Howard Y. (Howard Yuan-Hao)
Herschlag Daniel advisor. Thesis advisor