Characterization of T-Cell Lymphoblastic Leukemia Genetic Etiology

T-cell lymphoblastic leukemia (T-ALL) is inflict nearly 1500 children annually in the US. We recently reported using Sleeping Beauty mutagenesis as a forward genetics approach to model two major subtypes of T-ALL, typical and early T-cell precursor (ETP). The genetic mutations that define these subtypes is incomplete. Moreover, identifying driver from passenger mutations within these tumors is paramount for developing targeted therapeutics to specific genetic lesions.

T-ALL tumors were generated using a Cre-inducible Sleeping Beauty system to drive mutagenesis at either early (Vav-iCre) or late (CD4-Cre) stage of T-cell development. Synthetic gene standards were spiked into samples at known concentrations to permit quantitation of transposon insertion events within individual tumors. Samples were prepared by ligation mediated PCR and sequenced using an Illumina HiSeq. Using the standard as a barometer, common insertion sites (CIS) were defined as initiating, transition, or progression based on their abundance. We further developed a network analysis pathway to identify CISs within similar signaling axes.

Spiked standards were reproducibly seen at expected abundances in several replicated tumors. Further, we are able to distinguish insertions found at high abundances in tumors from lower occurring or background events. Together this allowed us to identify the order of mutations in each tumor and characterize mutations as initiators or progression events. Using our network analysis pathway we found Myc and Stat5b signaling axes driving the majority of ETP-ALL and Notch1 signaling driving typical T-ALL.

Our data are the first to utilize NextGen sequencing techniques to quantitate CIS events to understand the genetic etiology of T-ALL. Coupled with our network analysis software, we are able to more accurately identify driving pathways in individual T-ALL tumors induced by Sleeping Beauty mutagenesis.

No relevant conflicts of interest to declare.